Search method for suppressing the odor of sulfur-containing compounds

Abstract

To provide methods capable of searching for an inhibitor of the odor of sulfur-containing compounds, which have a sufficient odor inhibitory effect easily obtained in an actual product and are suitable for a practical deodorant.SOLUTION: Provided is a method for searching for an inhibitor of the odor of sulfur-containing compounds, comprising: mixing in the presence of metal ions at least one or more olfactory receptors selected from the group consisting of a polypeptide having a function equivalent to OR2T11, OR2T1, OR2T6, OR2T11, a polypeptide having a function equivalent to OR2T1, and a polypeptide having a function equivalent to OR2T6, and a test substance, and then bringing the olfactory receptors into contact with a sulfur-containing compound.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for searching for an agent for suppressing the odor of sulfur-containing compounds. [Background technology]

[0002] The sense of smell is recognized by the response of olfactory receptors present in olfactory nerve cells in the olfactory epithelium to odor molecules. It has been proposed to suppress the perception of malodors based on the response of olfactory receptors to odor molecules (Patent Document 1). Patent Document 1 discloses that a compound that acts as an agonist for olfactory receptors such as OR4S2 is selected as a methyl mercaptan odor inhibitor. The methyl mercaptan odor inhibitor in Patent Document 1 functions as an agonist for olfactory receptors such as OR4S2. Therefore, the methyl mercaptan odor inhibitor binds to OR4S2 and the like, desensitizing the olfactory receptors. As a result, olfactory sensitivity to malodors is reduced, and the perception of malodors is suppressed. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2020-10629 Summary of the Invention [Problem to be solved by the invention]

[0004] In the examples of Patent Document 1, an aqueous solution of methyl mercaptan sodium is directly added as a malodorous molecule to a liquid medium after culturing cells expressing olfactory receptors. However, the olfactory receptors in the olfactory epithelium of the nose actually respond to methyl mercaptan, a malodorous molecule floating in the air, dissolved in olfactory mucus. Thus, when an aqueous solution of methyl mercaptan sodium is directly added, a methyl mercaptan odor suppressant is being sought in an environment where the malodorous perception mechanism behaves differently from the actual behavior. Therefore, there is a possibility that the methyl mercaptan odor suppressant will not provide sufficient malodorous suppression effect in an actual product.

[0005] Furthermore, since the olfactory receptor agonist selected by the method of Patent Document 1 suppresses malodors by utilizing desensitization of olfactory receptors, it is necessary to maintain a high concentration of the agonist in a diffused state in the target space, which requires that the amount of diffusion and scattering of the agonist be set to be large, making it impractical as a deodorizer. Therefore, the present invention provides a method for searching for an inhibitor of the odor of sulfur-containing compounds that is suitable for practical deodorants and that can easily achieve a sufficient odor-inhibiting effect in actual products. [Means for solving the problem]

[0006] The present invention has the following aspects. [1] A method for searching for an inhibitor of the odor of sulfur-containing compounds, comprising mixing at least one olfactory receptor selected from the group consisting of OR2T11, OR2T1, OR2T6, polypeptides having a function equivalent to OR2T11, polypeptides having a function equivalent to OR2T1, and polypeptides having a function equivalent to OR2T6 with a test substance in the presence of metal ions, and then contacting the olfactory receptor with a sulfur-containing compound. [2] The method of searching for a sulfur-containing compound according to [1], wherein the sulfur-containing compound is in a gaseous state. [3] The screening method of [1] or [2], further comprising contacting the olfactory receptor with the sulfur-containing compound and then selecting a test substance that inhibits the response of the olfactory receptor as the inhibitor. [4] A method for searching for any of [1] to [3], wherein the amino acid sequence of a polypeptide having a function equivalent to that of OR2T11 shows 80% or more homology with the amino acid sequence of OR2T11. [5] A method for searching for any of [1] to [4], wherein the amino acid sequence of a polypeptide having a function equivalent to that of OR2T1 shows 80% or more homology with the amino acid sequence of OR2T1. [6] A method for searching for any of [1] to [5], wherein the amino acid sequence of a polypeptide having a function equivalent to that of OR2T6 shows 80% or more homology with the amino acid sequence of OR2T6. [7] The screening method according to any one of [1] to [6], wherein the sulfur-containing compound is at least one selected from the group consisting of hydrogen sulfide, methyl mercaptan, allyl mercaptan, dimethyl trisulfide, dimethyl disulfide, allyl disulfide, t-butyl mercaptan, 3-mercapto-3-methylbutan-1-ol, 4-mercapto-4-methyl-2-pentanone, methyl sulfide, and 2-propanethiol. [8] The screening method according to any one of [1] to [7], wherein the inhibitor is an antagonist of the olfactory receptor. [Effects of the Invention]

[0007] According to the present invention, a method is provided for searching for an inhibitor of the odor of sulfur-containing compounds that is suitable for practical deodorants and that can easily achieve a sufficient odor-inhibiting effect in actual products. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 2 shows the results of measuring the response strength (fold increase) of olfactory receptors to methyl mercaptan in an example. [Figure 2] FIG. 1 shows the results of measuring the response strength (fold increase) of olfactory receptors to hydrogen sulfide in an example. [Figure 3] FIG. 1 shows the results of measuring the response of OR2T11 to methyl mercaptan in an example. [Figure 4] FIG. 1 shows the results of measuring the response of OR2T1 to methyl mercaptan in an example. [Figure 5] FIG. 1 shows the results of measuring the response of OR2T6 to methyl mercaptan in an example. DETAILED DESCRIPTION OF THE INVENTION

[0009] As used herein, the following terms have the following meanings: A "polypeptide having a function equivalent to that of an olfactory receptor" refers to a polypeptide that can be expressed on a cell membrane and that, upon binding to an odorant molecule, induces the production of cAMP within the cell, or a polypeptide that promotes the influx of calcium ions from outside the cell into the cell. The term "sulfur-containing compound odor" refers to the odor generated by compounds containing sulfur atoms in the molecule. The symbol "to" indicating a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0010] The sequence identity of an amino acid sequence can be determined, for example, as the sequence identity of a subject amino acid sequence to a reference amino acid sequence, as follows: First, the reference amino acid sequence and the subject amino acid sequence are aligned. Here, gaps may be included in each amino acid sequence to maximize sequence identity. Next, the number of amino acid residues of matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be determined according to the following formula (1): Sequence identity (%) = (number of matching amino acid residues / total number of amino acid residues in the target amino acid sequence) × 100 Equation (1)

[0011] <Method for searching for an agent for suppressing the odor of sulfur-containing compounds> In one embodiment of the method for searching for an inhibitor of the odor of sulfur-containing compounds, at least one olfactory receptor selected from the group consisting of OR2T11, OR2T1, OR2T6, polypeptides having a function equivalent to OR2T11, polypeptides having a function equivalent to OR2T1, and polypeptides having a function equivalent to OR2T6 is mixed with a test substance in the presence of metal ions, and then the olfactory receptor is brought into contact with a gaseous sulfur-containing compound.

[0012] If the olfactory receptor mixed with the test substance does not elicit a response from the test substance, and if the olfactory receptor becomes less responsive to the gaseous sulfur-containing compound when it comes into contact with the gaseous sulfur-containing compound, the test substance mixed with the specific olfactory receptor can be said to function as an antagonist. In this case, the test substance can be expected to be useful as an agent for suppressing the odor of sulfur-containing compounds. Therefore, in one embodiment, a test substance that suppresses the response of an olfactory receptor when the olfactory receptor is contacted with a gaseous sulfur-containing compound can be selected as an inhibitor of the odor of a sulfur-containing compound. In other words, in one embodiment, the inhibitor of the odor of a sulfur-containing compound can also be said to be an antagonist of the olfactory receptor.

[0013] OR2T11 is an olfactory receptor whose expression in human olfactory receptor cells has been confirmed, and is registered with GenBank (NCBI) under Gene ID: 127077. OR2T11 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. In one embodiment, OR2T11 can be replaced by a polypeptide having a function equivalent to OR2T11. The amino acid sequence of the polypeptide having a function equivalent to OR2T11 preferably exhibits 80% or more homology with the amino acid sequence of OR2T1, more preferably 85% or more homology, even more preferably 90% or more homology, even more preferably 95% or more homology, particularly preferably 98% or more homology, and most preferably 99% or more homology.

[0014] OR2T1 is an olfactory receptor whose expression in human olfactory receptor cells has been confirmed, and is registered with GenBank (NCBI) under Gene ID: 26696. OR2T1 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. In one embodiment, OR2T1 can be replaced by a polypeptide having a function equivalent to OR2T1. The amino acid sequence of the polypeptide having a function equivalent to OR2T1 preferably exhibits 80% or more homology with the amino acid sequence of OR2T1, more preferably 85% or more homology, even more preferably 90% or more homology, even more preferably 95% or more homology, particularly preferably 98% or more homology, and most preferably 99% or more homology.

[0015] OR2T6 is an olfactory receptor whose expression in human olfactory receptor cells has been confirmed, and is registered with GenBank (NCBI) under Gene ID: 254879. OR2T6 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. In one embodiment, OR2T6 can be replaced by a polypeptide having a function equivalent to OR2T6. The amino acid sequence of the polypeptide having a function equivalent to OR2T6 preferably exhibits 80% or more homology with the amino acid sequence of OR2T6, more preferably 85% or more homology, even more preferably 90% or more homology, even more preferably 95% or more homology, particularly preferably 98% or more homology, and most preferably 99% or more homology.

[0016] Other polypeptides having functions equivalent to those of OR2T11, OR2T1, and OR2T6 include homologous olfactory receptors derived from animals other than humans, such as mice, rats, and other experimental model organisms.

[0017] The olfactory receptor can be used in any manner as long as it does not lose its responsiveness to sulfur-containing compounds. For example, the olfactory receptor can be used in the form of cells or tissues naturally expressing the olfactory receptor and their cultures; membranes of olfactory receptor cells carrying the olfactory receptor; genetically modified cells expressing the olfactory receptor and their cultures; membranes of genetically modified cells expressing the olfactory receptor; lipid bilayer membranes expressing the olfactory receptor, etc. Furthermore, tissues having olfactory mucus (such as the olfactory epithelium and the olfactory mucosa) may also be used as the olfactory receptor.

[0018] In one embodiment, the olfactory receptor is preferably a cell that naturally expresses an olfactory receptor, a genetically modified cell that expresses an olfactory receptor, or a culture thereof. In particular, the use of human-derived genetically modified cells that express an olfactory receptor is preferred. Human-derived genetically modified cells can be prepared, for example, by transforming cultured human cells with a vector incorporating a gene encoding an olfactory receptor. During transformation, in order to promote the expression of the olfactory receptor in the cell membrane, it is preferable to use a gene encoding an RTP (receptor-transporting protein) in addition to the gene encoding the olfactory receptor. This is because the gene encoding human RTP1S can be introduced into cells together with the gene encoding the olfactory receptor. An example of RTP1S is human RTP1S, which is registered in GenBank as GeneID: 132112.

[0019] The test substance is not particularly limited as long as it is a substance desired to be used as an inhibitor of the odor of sulfur-containing compounds. The test substance may be a naturally occurring substance or a synthetic substance. Furthermore, the test substance may be a single substance or a mixture containing two or more substances. For example, a fragrance of a single substance may be used, or a fragrance blended with multiple substances may be used. Considering the possibility of commercializing the test substance as a deodorant, it is preferable that the test substance have an odor that is different from the odor of the sulfur-containing compound, and is preferably a volatile substance. It is also preferable to use a substance with a low odor intensity. The method for mixing the test substance is not particularly limited. The test substance may be mixed into the culture medium for cells expressing the olfactory receptor, or the test substance may be dripped, sprayed, or sprayed onto cells or tissues expressing the olfactory receptor.

[0020] Examples of metal ions include copper ions and silver ions, with copper ions being preferred. The concentration of the metal ions is not particularly limited, and may be 10 to 300 μM or 1 to 1000 μM. The manner in which the metal ions are used and present is not particularly limited. Examples include a method of mixing an olfactory receptor with a test substance in a metal ion-containing solution; a method of mixing a membrane carrying an olfactory receptor or cells or tissues in which an olfactory receptor is expressed with a test substance while immersed in a metal ion-containing solution; a method of adding a metal ion to a culture medium in which cells or tissues in which an olfactory receptor is expressed are cultured, and then adding the test substance; and a method of mixing a metal ion-containing solution together with a test substance with a culture medium in which cells or tissues in which an olfactory receptor is expressed are cultured.

[0021] In one embodiment, the olfactory receptor and the test substance are mixed in the presence of metal ions, and then the response of the olfactory receptor is measured before contact with the gaseous sulfur-containing compound. For example, data obtained by measuring the response of the olfactory receptor immediately after mixing the olfactory receptor and the test substance may be used as reference data and may be used in conjunction with the test data described below. The specific measurement method is not particularly limited as long as it can evaluate the response of the olfactory receptor. For example, the amount of intracellular cAMP can be measured. The response of the olfactory receptor can be evaluated by using the amount of intracellular cAMP as an indicator of the response of the olfactory receptor. Examples of methods for measuring the amount of intracellular cAMP include ELISA and reporter gene assay. Other examples include calcium imaging and electrophysiological measurements. In electrophysiological measurements, for example, test cells (e.g., Xenopus oocytes) are prepared in which an olfactory receptor is co-expressed with other ion channels, and the action potential of the ion channel on the test cell may be measured using the patch clamp method, two-electrode voltage clamp method, or the like.

[0022] The sulfur-containing compound is a compound containing a sulfur atom in the molecule. The sulfur-containing compound is not particularly limited, but examples thereof include inorganic sulfur compounds such as hydrogen sulfide and sulfur dioxide; sulfanyl alkanols such as 2-mercapto-3-methyl-1-butanol, 3-mercapto-2-methyl-1-pentanol, 3-mercapto-2-methyl-1-propanol, 3-mercapto-3-methyl-1-butanol, 3-mercapto-1-hexanol, 3-methyl-3(2-methyldisulfanyl)-butan-1-ol, and 3-mercapto-3-methyl-1-hexanol; sulfanyl aldehydes and sulfanyl ketones such as 1-mercapto-2-methylpentanal, 1-mercapto-3-pentanone, 2-mercapto-3-pentanone, 3-mercapto-3-pentanone, and 4-mercapto-4-methyl-2-pentanone; sulfanyl esters such as 3-methyl-3-mercaptobutyl acetate and 3-methyl-3-mercaptobutyl formate; methyl mercaptan, ethyl mercaptan, allyl mercaptan, t-butyl mercaptan, and 2-methylpropyl mercaptan; Thiols such as 1-methoxyheptane-3-thiol, 4-methoxy-2-methylbutane-2-thiol, 1-propanethiol, 2-propanethiol, 2-sulfanylethanol, 2-(methylthio)-2-propanethiol, and 3-mercapto-3-methylbutan-1-ol; isothiocyanates such as methyl isothiocyanate, ethyl isothiocyanate, and allyl isothiocyanate; methylthiomethane, methyldithiomethane, methyltrithiomethane, ethylthioethane, and ethyldithio Thiomethanes and other thiomethanes; thiophenes such as thiophene, dithiophene, terthiophene, tetrahydrothiophene, 2,5-dimethylthiophene, and 2-acetylthiophene; thiazoles such as 2-isobutylthiazole, 2-acetylthiazole, and 4-ethyl-5-propylthiazole; thiolanes such as 3,5-dimethyl-1,2,4-trithiolane, 1,2,4-trithiolane, and 3-methyl-1,3,5-trithiolane; thiocarboxylic acids such as thioglycolic acid and dithioglycolic acid;Examples of sulfides include methyl-2-propenyl disulfide, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, diallyl sulfide, diallyl disulfide, diallyl trisulfide, diallyl tetrasulfide, thiirane, (1-methylethyl)-thiirane, and diphenyl disulfide; allicin; alliin; ajoene; lenthionine; cysteine; glutathione; methylthiomethane; thioacetic acid; methyl thioacetate; and the like. The sulfur-containing compounds may be used alone or in combination of two or more. In addition, a complex odor obtained by combining the sulfur-containing compounds with malodorous substances other than the sulfur-containing compounds, or an odor collected from any space containing the sulfur-containing compounds may also be used.

[0023] In one embodiment, the olfactory receptor is contacted with a gaseous sulfur-containing compound, and then the response of the olfactory receptor is measured. For example, data measuring the response of the olfactory receptor after contact with a gaseous sulfur-containing compound may be used as test data. If the response of the olfactory receptor to the test substance does not change in the reference data with an increase in the amount of test substance added, and the response of the olfactory receptor to the sulfur-containing compound decreases after contact with the gaseous sulfur-containing compound in the test data, then the test substance mixed with a specific olfactory receptor functions as an antagonist. In this case, the test substance is considered to be useful as an inhibitor of the odor of sulfur-containing compounds. In one embodiment, after mixing the olfactory receptor with the test substance, the test substance that inhibits the contact response of the gaseous sulfur-containing compound to the olfactory receptor can be selected as an inhibitor of the odor of sulfur-containing compounds.

[0024] In one embodiment, the method includes measuring the response of the olfactory receptors in a test group mixed with the test substance and in a control group, and comparing the respective measurement results. By comparing the response of the olfactory receptors in a test group mixed with the test substance with the response in the control group, for example, the inhibitory effect of the test substance on the olfactory receptors against the odor of sulfur-containing compounds can be evaluated. Examples of control groups include olfactory receptors that have not been mixed with a test substance; olfactory receptors that have been mixed with a test substance but have not been contacted with a gaseous sulfur-containing compound; olfactory receptors that have been mixed with a relatively low concentration of a test substance; olfactory receptors before the addition of the test substance; and cells in which no olfactory receptors are expressed.

[0025] In one embodiment, for example, if the response in the test group using the test substance is suppressed compared to the control group, the test substance can be selected as an inhibitor of the odor of sulfur-containing compounds, i.e., an antagonist of the olfactory receptor. For example, if the response index of the olfactory receptor in the test group is statistically significantly reduced compared to the control group, the test substance can be selected as an inhibitor of the odor of sulfur-containing compounds.

[0026] The manner of contact between the olfactory receptor mixed with the test substance and the sulfur-containing compound is not particularly limited, as long as the gaseous sulfur-containing compound can contact the olfactory receptor. Examples include a method in which a membrane carrying an olfactory receptor or a cell or tissue expressing an olfactory receptor is placed in a sealed container containing the gaseous sulfur-containing compound; or a method in which a membrane carrying an olfactory receptor or a cell or tissue expressing an olfactory receptor is placed in a sealed container, and then the gaseous sulfur-containing compound is supplied into the sealed container. When contacting the olfactory receptor with the sulfur-containing compound, a culture plate, a petri dish, or a circulator may be used.

[0027] In one embodiment, a cell culture plate having a plurality of wells may be used. For example, cells or tissues expressing olfactory receptors and a test substance are dispensed into each of the plurality of wells of the cell culture plate and mixed, and a gaseous sulfur-containing compound is supplied around the cell culture plate to bring the sulfur-containing compound into contact with the olfactory receptors. By mixing the test substance with the olfactory receptors in advance in this way, the olfactory receptors in each well can be brought into contact with the gaseous sulfur-containing compound, so that the response start time of the olfactory receptors in each well to the malodorous odor can be synchronized. Therefore, there is less of a time lag between the wells at the start of the response, which occurs when a malodorous substance is added to the olfactory receptors in each well one by one in advance and then the olfactory receptor response is measured. Therefore, according to one embodiment, it is possible to measure olfactory receptor response data with less measurement error. When a cell culture plate having multiple wells is used, the olfactory receptors in each well may be the same or different from each other.

[0028] In one embodiment, a sensory test may be further performed. For example, a test substance expected to be useful as an inhibitor of the odor of sulfur-containing compounds is selected as a candidate substance for the inhibitor of the odor of sulfur-containing compounds, and the inhibitory effect of the candidate substance on the odor of sulfur-containing compounds is evaluated by a sensory test. A candidate substance that is confirmed to have an inhibitory effect on the odor of sulfur-containing compounds in the sensory test may be selected as an inhibitor of the odor of sulfur-containing compounds.

[0029] The sensory test can be performed according to a normal evaluation procedure for deodorants. For example, the evaluator may smell the odor of the sulfur-containing compound simultaneously with the odor of the candidate substance and evaluate the intensity of the odor of the sulfur-containing compound, or may smell the odor of the sulfur-containing compound separately from the odor of the candidate substance and evaluate the intensity of the odor of the sulfur-containing compound. The evaluation results obtained are compared with the intensity of the sulfur-containing compound odor alone. Candidate substances that are evaluated as reducing the intensity of the sulfur-containing compound odor as a result of the sensory test are expected to be useful as suppressants for the sulfur-containing compound odor.

[0030] (Application) The suppressant for the odor of sulfur-containing compounds can be used as an active ingredient of a deodorizer for the odor of sulfur-containing compounds. Specific usage modes are not particularly limited. For example, the suppressant for the odor of sulfur-containing compounds can be used as an active ingredient of a composition or article for suppressing the odor of sulfur-containing compounds, or can be used in the manufacture of a composition or article for suppressing the odor of sulfur-containing compounds. Application examples include human and animal toilets or excrement treatment; excrement treatment in medical facilities, nursing homes, and care facilities; disposable diapers, sanitary products; permanents; beauty salons and the like that use permanents; clothing such as underwear, undergarments, masks, face shields, and linens, cloth products, and textiles; laundry detergents, fabric softeners; topical preparations such as cosmetics, cleaning agents, and deodorants, pharmaceuticals; foods, etc.; and manufacturing equipment for products that emit sulfur-containing compound odors. However, the applications of the inhibitor for the odor of sulfur-containing compounds are not limited to these examples.

[0031] (Action and effect) In the screening method according to one embodiment described above, an olfactory receptor and a test substance are mixed in the presence of metal ions, and then the olfactory receptor is brought into contact with a gaseous sulfur-containing compound to test whether the test substance functions as an inhibitor of the odor of the sulfur-containing compound, i.e., as an antagonist of the olfactory receptor. By contacting olfactory receptors mixed with a test substance in the presence of metal ions with gaseous sulfur-containing compounds, it is possible to reproduce the actual response of olfactory receptors in the olfactory epithelium to gaseous sulfur-containing compounds floating in the air. As a result, it is possible to search for inhibitors of sulfur-containing compound odors while approximating the perception mechanism of sulfur-containing compound odors to the actual response of olfactory receptors in the nose. It is believed that inhibitors of sulfur-containing compound odors identified in this way will have sufficient deodorizing effects when used in actual products. Furthermore, because the inhibitor of the odor of sulfur-containing compounds is an antagonist of the olfactory receptor, the concentration required to obtain the same level of deodorizing effect in the target space can likely be set lower when an antagonist is used than when an agonist is used. Therefore, the inhibitor of the odor of sulfur-containing compounds is practical as an active ingredient in deodorizers for sulfur-containing compound odors.

[0032] According to one embodiment of the screening method, a gaseous sulfur-containing compound is used, which allows the reaction of olfactory receptors in the nose to be reproduced and allows the screening of olfactory receptor antagonists that respond to the odor of a sulfur-containing compound. Furthermore, since the sulfur-containing compound that causes the malodor is supplied as a gas, it does not need to be a single compound; for example, a complex odor such as air collected from a malodorous space can be used as the gaseous sulfur-containing compound odor.

[0033] According to one embodiment of the screening method, an inhibitor of the odor of sulfur-containing compounds, i.e., an olfactory receptor antagonist, can be used as an active ingredient in a deodorant. Because antagonists antagonize the odor of sulfur-containing compounds, inhibiting the response of olfactory receptors and the perception of malodors, there is no need to maintain a pre-dispersed state like with agonists. Therefore, antagonist inhibitors of the odor of sulfur-containing compounds are also suitable for instant deodorizing applications such as sprays. [Example]

[0034] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0035] <Preparation of human olfactory receptor expressing cells> (pCI-human olfactory receptor vector, pCI-human RTP1S vector) Based on the sequence information registered in GenBank, the genes encoding the human olfactory receptors listed in Tables 1 and 2 were cloned. Each gene was cloned by PCR using human genomic DNA Human mixed (G3041: Promega) as a template. Each gene amplified by PCR was inserted into a pCI vector (Invitrogen) according to the manufacturer's protocol. Specifically, a Rho tag sequence was inserted using the NheI and BamHI restriction enzyme sites present in the pCI vector, and the olfactory receptor gene was inserted downstream of the Rho tag sequence using the downstream Mlu and NotI restriction enzyme sites. Next, the gene encoding human RTP1S was inserted into the MluI and NotI restriction enzyme sites of the pCI vector.

[0036] [Table 1]

[0037] [Table 2]

[0038] (olfactory receptor-expressing cells) Hana3A cells were cultured in a 96-well plate (Corning, BioCoat) until they reached 50% confluence. A reaction solution with the composition shown in Table 3 was prepared and left to stand in a clean bench for 15 minutes. Then, 50 μL of the solution was added to each well of the 96-well plate (Corning, BioCoat). The cells were cultured for 24 hours in an incubator maintained at 37°C and a 5% CO2 atmosphere, resulting in Hana3A cells expressing each of the 392 human olfactory receptors.

[0039] [Table 3]

[0040] <Sulfur-containing compounds> The following odorants were used as sulfur-containing compounds: Methyl mercaptan (2% gas / nitrogen) (Sogo Sangyo Co., Ltd.) Hydrogen sulfide (1.5% gas / nitrogen) (Sogo Sangyo Co., Ltd.) Allyl mercaptan Dimethyl trisulfide Dimethyl disulfide Allyl disulfide t-Butyl mercaptan 3-Mercapto-3-methylbutan-1-ol 4-Mercapto-4-methyl-2-pentanone Methyl sulfide 2-Propanethiol

[0041] <Glo Sensorアッセイ> The Glo Sensor assay was used to measure the response of olfactory receptors. Olfactory receptors expressed in Hana3A cells couple to endogenous Gαs and Gαlf, activating adenylate cyclase and increasing intracellular cAMP levels. The increase in intracellular cAMP was measured as luminescence from the firefly luciferase gene, and the response of the olfactory receptors was measured. Luciferase activity was measured using Glo Sensor cAMP Reagent (Promega) according to the product protocol. For each stimulation condition, the luminescence value derived from luciferase before odor stimulation was divided by the luminescence value derived from luciferase after odor stimulation to calculate (luminescence value after stimulation) / (luminescence value before stimulation). The response intensity induced by odorant stimulation was measured as (luminescence value after stimulation) / (luminescence value before stimulation).

[0042] <Search for olfactory receptors that respond to sulfur-containing compounds> (Identification of OR2T11, OR2T1, and OR2T6) The medium was removed from the culture of olfactory receptor-expressing cells, and 25 μL of Glo Sensor cAMP Reagent diluted in HBSS buffer containing 10 mM HEPES was added to each well of a 96-well plate. The cells were cultured in a light-protected environment for 2–3 hours to allow intracellular introduction of the cAMP Reagent. After this, the 96-well plate and an air circulator were placed in a 5 L Flex Sampler bag and filled with pure air. Finally, methyl mercaptan gas or hydrogen sulfide gas, as odor molecules, was injected using a syringe to achieve the desired final gas-phase concentration. The odor molecules were allowed to contact the olfactory receptor-expressing cells for 10 minutes, after which a Glo Sensor assay was performed to measure the fold increase in the response of the olfactory receptor to the odor molecules. The measurement results for hydrogen sulfide (40 ppm concentration) and methyl mercaptan (7 ppm concentration) are shown in Figure 1 and Figure 2, respectively. The results are expressed as the relative response intensity to odor stimuli in each receptor-expressing cell in the presence of copper ions, with the response intensity in the absence of odor stimuli set to 1 (Figures 1 and 2). The response of cells expressing each of the 392 types of olfactory receptors to odor molecules was measured in the presence of copper ions, and OR2T11 was identified as the olfactory receptor that showed the highest response to methyl mercaptan. OR2T1 was also identified as the olfactory receptor that showed the highest response to hydrogen sulfide. OR2T1 and OR2T6 were also identified as olfactory receptors that showed the highest response to hydrogen sulfide.

[0043] (Dependence of responses of OR2T11, OR2T1, and OR2T6 on sulfur-containing compounds) The responses of OR2T11, OR2T1, and OR2T6 to different concentrations of methyl mercaptan were measured. The results for OR2T11 are shown in Figure 3, those for OR2T1 in Figure 4, and those for OR2T6 in Figure 5. As a result, OR2T11 and OR2T1 showed a concentration-dependent response to methyl mercaptan, confirming that they are methyl mercaptan receptors. Of these, OR2T11 was found to be the most responsive and particularly sensitive receptor to methyl mercaptan (Figures 3-5).

[0044] (Confirmation of OR2T11 response to sulfur-containing compounds) The response of OR2T11 to allyl mercaptan, dimethyl trisulfide, dimethyl disulfide, allyl disulfide, t-butyl mercaptan, 3-mercapto-3-methylbutan-1-ol, 4-mercapto-4-methyl-2-pentanone, methyl sulfide, and 2-propanethiol at a concentration of 10 μM was measured. The results are shown in Table 4. As a result, OR2T11 responded to these sulfur-containing compounds, confirming that it is a receptor for these sulfur-containing compounds.

[0045] [Table 4]

[0046] <Test substance> As test substances, 105 compounds were diluted with HBSS buffer containing 10 mM HEPES and prepared to a final concentration of 200 μM (initial concentration 100 μM).

[0047] <Search for Antagonists of OR2T11> The medium was removed from the cultures of OR2T11-expressing cells, and 25 μL of GloSensor cAMP Reagent diluted with HBSS buffer containing 10 mM HEPES was added to each well of a 96-well plate. The cells were cultured for 2 - 3 hours to introduce the cAMP Reagent into the cells. Then, a GloSensor assay was performed to measure the response intensity of the olfactory receptor before adding the test substance and obtain reference data. Subsequently, 25 μL of the test substance was added to each well of the 96-well plate, and after 10 minutes, a GloSensor assay was performed to measure the response intensity (fold increase) of the olfactory receptor to the test substance and obtain test data. Subsequently, a 96-well plate and a fan for air circulation were placed in a 5 L Flexsample bag and filled with pure air. Finally, methyl mercaptan gas or hydrogen sulfide gas as malodorous molecules was injected with a syringe so that the gas-phase final concentration reached a predetermined value. The olfactory receptor-expressing cells were contacted with the malodorous molecules for 10 minutes, and then a GloSensor assay was performed to measure the response intensity (fold increase) of the olfactory receptor to the malodorous molecules. As a result of measuring the response intensity, geraniol, citral, linalyl acetate, terpinyl acetate, acetyl cedrene, β-ionone, α-ionone, α-isomethyl ionone, α-damascone, δ-damascone, β-damascenone, β-damascone, and α-iron were identified as antagonists. The measurement results of the response intensity for these 13 antagonists are shown in Table 5.

[0048]

Table 5

[0049] As shown in Table 5, 13 compounds suppressed the response of OR2T11 to methyl mercaptan, and are therefore considered to function as antagonists of OR2T11.

[0050] The response strength of the above 13 antagonists was measured using hydrogen sulfide as a stimulus in the same manner as in the case of methyl mercaptan. The results are shown in Table 6.

[0051] [Table 6]

[0052] As shown in Table 6, 13 compounds also suppressed the response of OR2T11 to hydrogen sulfide, suggesting that they function as antagonists of OR2T11. [Industrial Applicability]

[0053] According to the present invention, it is possible to search for an inhibitor of the odor of sulfur-containing compounds that is suitable for practical deodorants and that can easily achieve a sufficient odor-inhibiting effect in actual products.

Claims

1. The method comprises mixing at least one olfactory receptor selected from the group consisting of OR2T6 and polypeptides having an amino acid sequence that is 90% or more homologous to the amino acid sequence of OR2T6 with a test substance in the presence of copper ions, and then contacting the olfactory receptor with a gaseous sulfur-containing compound, and then measuring the response of the olfactory receptor by Glo Sensor assay; The sulfur-containing compound is hydrogen sulfide. A method for discovering antagonists of olfactory receptors for sulfur-containing compound odors.

2. The method for discovering an antagonist of an olfactory receptor for the odor of a sulfur-containing compound described in claim 1 further comprises contacting the olfactory receptor with the sulfur-containing compound and then selecting a test substance that inhibits the response of the olfactory receptor as the antagonist.

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