Searching for an agent to suppress aldehyde odor

Abstract

To provide a method for obtaining an inhibitor capable of sufficiently exhibiting deodorization effect on aldehyde odor.SOLUTION: A search method of aldehyde odor inhibitor includes mixing at least one or more olfactory receptors selected from a group consisting of OR6B1, Olfr449, polypeptides having a function equivalent to OR6B1, and a polypeptide having a function equivalent to Olfr449, with a test substance.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for searching for an agent for suppressing aldehyde odor. [Background technology]

[0002] Aldehydes are contained in factory exhaust, cigarette smoke, body odor, bad breath, etc., and produce unpleasant odors. Regarding the deodorization of aldehyde odors, physical deodorization using adsorption treatment and chemical deodorization using neutralization reactions have been proposed. For example, Patent Document 1 discloses an aldehyde adsorbent in which a compound having an amino group or an imino group is supported within the pores of a porous silica material.

[0003] On the other hand, the sense of smell is recognized by olfactory receptors in olfactory nerve cells, which respond to odor molecules. Focusing on the response of these olfactory receptors to odor molecules, it has been proposed to suppress the perception of malodors. For example, Patent Document 2 discloses receptors that recognize a wide range of odorants indiscriminately, i.e., broadly tuned olfactory receptors, primarily OR2W1, OR1A1, and OR10A6. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-82786 [Patent Document 2] Japanese Patent Application Publication No. 2018-121623 Summary of the Invention [Problem to be solved by the invention]

[0005] In physical and chemical deodorization such as that described in Patent Document 1, the odor is eliminated by converting the aldehyde that causes the odor into another compound using a deodorizer or adsorbent. This requires molecular contact between the aldehyde and the deodorizer, etc. Considering this point, for example, while the deodorizing effect on aldehyde odors can be achieved over time in an enclosed space, it is difficult to achieve the deodorizing effect in an open space or a large space. Therefore, the inventors conducted extensive research and came up with the idea of ​​suppressing the response of olfactory receptors that are sensitive to aldehyde odors, in order to more easily obtain a deodorizing effect against aldehyde odors under various conditions of use, regardless of the usage environment. However, the broadly tuned olfactory receptors disclosed in Patent Document 2 lack sufficient specificity in their response to aldehyde odors. Therefore, simply suppressing the response of these broadly tuned olfactory receptors does not fully exert the deodorizing effect on aldehyde odors.

[0006] The present invention provides a method for obtaining an inhibitor that is likely to exhibit a sufficient deodorizing effect against aldehyde odors. [Means for solving the problem]

[0007] The present invention has the following aspects. [1] A method for searching for an inhibitor of an olfactory receptor, the method comprising mixing a test substance with at least one olfactory receptor selected from the group consisting of OR6B1, Olfr449, a polypeptide having a function equivalent to OR6B1, and a polypeptide having a function equivalent to Olfr449. [2] The screening method of [1], further comprising contacting the olfactory receptor with a lower aldehyde after mixing the olfactory receptor with a test substance. [3] The discovery method of [2] further comprises contacting the olfactory receptor with the lower aldehyde and then selecting a test substance that inhibits the response of the olfactory receptor as an inhibitor of aldehyde odor. [4] The method for screening according to [2] or [3], wherein the lower aldehyde is at least one selected from the group consisting of acetaldehyde, propanal, butanal, and pentanal. [5] A search method according to any of [1] to [4], in which the amino acid sequence of a polypeptide having a function equivalent to that of OR6B1 shows 80% or more homology with the amino acid sequence of OR6B1. [6] A method for searching for any of [1] to [5], wherein the amino acid sequence of a polypeptide having a function equivalent to Olfr449 shows 80% or more homology with the amino acid sequence of Olfr449. [7] The screening method according to any one of [1] to [6], wherein the inhibitor is an antagonist of the olfactory receptor. [Effects of the Invention]

[0008] According to the present invention, it is possible to obtain an inhibitor that is likely to exhibit a sufficient deodorizing effect against aldehyde odors. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the results of measuring the response strength (fold increase) of olfactory receptors to acetaldehyde in an example. [Figure 2] This figure shows the results of measuring the concentration dependence of the response intensity of OR6B1 to acetaldehyde in an example. [Figure 3] This figure shows the results of measuring the response strength (fold increase) of OR6B1 to lower aldehydes and aldehydes with 6 or more carbon atoms in an example. DETAILED DESCRIPTION OF THE INVENTION

[0010] 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 promotes the influx of calcium ions from outside the cell into the cell. The symbol "to" indicating a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0011] The sequence identity or homology of an amino acid sequence can be determined 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 the 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)

[0012] (Mixture of test substance and olfactory receptor) In one embodiment, a method for searching for an inhibitor of aldehyde odor involves mixing a test substance with at least one olfactory receptor selected from the group consisting of OR6B1, Olfr449, a polypeptide having a function equivalent to OR6B1, and a polypeptide having a function equivalent to Olfr449. Hereinafter, in this specification, at least one or more olfactory receptors selected from the group consisting of OR6B1, Olfr449, polypeptides having functions equivalent to OR6B1, and polypeptides having functions equivalent to Olfr449 may be referred to as "specific olfactory receptors."

[0013] OR6B1 is an olfactory receptor whose expression in human olfactory neurons has been confirmed, and is registered with GenBank (NCBI) under Gene ID: 135946. OR6B1 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0014] Olfr449 is an olfactory receptor whose expression in mouse olfactory neurons has been confirmed, and is registered with GenBank (NCBI) under Gene ID: 259067. Olfr449 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0015] As a result of extensive research, the present inventors have found that specific olfactory receptors, such as OR6B1 and Olfr449, specifically respond to lower aldehydes. Therefore, it is believed that the perception of aldehyde odors can be effectively suppressed by inhibiting the response of these specific olfactory receptors.

[0016] In one embodiment of the screening method, a test substance is mixed with specific olfactory receptors. These specific olfactory receptors respond specifically to lower aldehydes, which are the main cause of aldehyde odors. Therefore, if the test substance inhibits the response of the specific olfactory receptors as a result of mixing the specific olfactory receptors with the test substance, the test substance is likely to exhibit a sufficient deodorizing effect against aldehyde odors. Therefore, according to the searching method of one embodiment, it is possible to obtain an inhibitor that is likely to exhibit a sufficient deodorizing effect against aldehyde odors.

[0017] The test substance is a substance desired to be used as an aldehyde odor suppressant. Therefore, the test substance is not particularly limited. Furthermore, the test substance may be a naturally occurring substance or a synthetic substance.

[0018] Considering the commercialization of an aldehyde odor inhibitor as a deodorant, the test substance is preferably a substance that gives off a different odor from the aldehyde odor, and more preferably a volatile substance. It is also preferable to use an odorless test substance or a test substance with a low odor intensity.

[0019] The test substance may be a single substance or a mixture containing two or more substances. For example, a single fragrance substance or a blend of fragrance substances may be used.

[0020] 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.

[0021] When the olfactory receptor and the test substance are mixed, metal ions may also be mixed. 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 a 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.

[0022] 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.

[0023] (Contact with lower aldehydes) In one embodiment of the screening method, a specific olfactory receptor is mixed with a test substance, and then the olfactory receptor is contacted with a lower aldehyde. If the response to the aldehyde odor at the specific olfactory receptor after mixing with the test substance is relatively suppressed compared to the case in the absence of the test substance, the test substance is considered to be useful as an inhibitor of aldehyde odor.

[0024] If the following two conditions are satisfied at the olfactory receptor after mixing with the test substance, the test substance can be considered to function as an antagonist. After mixing with the test substance, the olfactory receptors are less likely to respond to lower aldehydes when they come into contact with the lower aldehyde than when they are not mixed with the test substance (i.e., in the absence of the test substance). -Olfactory receptors do not respond to the test substance after mixing with the test substance.

[0025] When the test substance functions as an antagonist, it can be expected to be useful as an agent for suppressing aldehyde odor. In one embodiment, a test substance that inhibits the response of an olfactory receptor to a lower aldehyde when the olfactory receptor is contacted with the lower aldehyde can be selected as an aldehyde odor inhibitor. Therefore, in one embodiment, the aldehyde odor inhibitor can also be said to be an olfactory receptor antagonist.

[0026] Lower aldehydes have an aldehyde group and have 1 to 10 carbon atoms. 4 The lower aldehyde is not particularly limited, and may be a straight-chain compound or a branched-chain compound. The lower aldehyde may also be a compound having a cyclic structure. The lower aldehydes may be used alone or in combination of two or more.

[0027] Examples of lower aldehydes include linear aldehydes such as formaldehyde, acetaldehyde, propanal, butanal, and pentanal; branched aldehydes such as 2-methylpropanal and 2-methylbutanal; and cyclic aldehydes such as cyclopropanecarboxaldehyde. In particular, specific olfactory receptors such as OR6B1 tend to exhibit specific response strength to at least one or more lower aldehydes selected from the group consisting of acetaldehyde, propanal, butanal, and pentanal.

[0028] The manner of contacting the olfactory receptor mixed with the test substance with the lower aldehyde is not particularly limited. For example, a method of placing a membrane carrying an olfactory receptor or a cell or tissue expressing an olfactory receptor in a sealed container containing a lower aldehyde; or a method of placing a membrane carrying an olfactory receptor or a cell or tissue expressing an olfactory receptor in a sealed container and then supplying the lower aldehyde into the sealed container. Alternatively, a liquid lower aldehyde may be vaporized in a sealed container. When the olfactory receptor is brought into contact with the lower aldehyde, a culture plate, a petri dish, or a circulator may be used.

[0029] When the lower aldehyde is brought into contact with the olfactory receptor, the lower aldehyde may be in a gaseous state, a liquid state, or a solid state. In terms of reproducing the behavior of actual olfactory perception, the use of a gaseous lower aldehyde is preferred, but is not necessarily limited to a gas. A complex odor in which a malodorous substance other than a lower aldehyde is combined, or an odor collected from any space containing a lower aldehyde may also be used.

[0030] According to one embodiment of the screening method, gaseous lower aldehydes can be 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 aldehyde odors. Furthermore, because the lower aldehydes that cause malodors are supplied as gases, they do not need to be single compounds; for example, a complex odor such as air collected from a malodorous space can be used as the gaseous aldehyde odor.

[0031] In one embodiment of the screening method, it is preferable to contact olfactory receptors mixed with a test substance in the presence of metal ions with a gaseous lower aldehyde. This is because it is possible to reproduce the actual response of olfactory receptors in the olfactory epithelium to gaseous lower aldehydes floating in the air. In this case, the mechanism of perception of aldehyde odors can be made closer to the actual response of olfactory receptors in the nose, allowing for the screening of aldehyde odor inhibitors. It is believed that aldehyde odor inhibitors identified in this way are likely to exhibit deodorizing effects when actually used in products such as deodorants.

[0032] In one embodiment, the olfactory receptor response may be measured after mixing the olfactory receptor with the test substance and before contacting with the lower aldehyde. For example, data obtained by measuring the olfactory receptor response immediately after mixing the olfactory receptor with the test substance may be used as reference data and compared with the test data described below.

[0033] In one embodiment, the olfactory receptor mixed with the test substance may be contacted with a lower aldehyde, and then the response of the olfactory receptor may be measured. Data obtained by measuring the response of the olfactory receptor after contact with the lower aldehyde can be used as test data.

[0034] In one embodiment, the responses of the olfactory receptors in a test group, i.e., a plurality of olfactory receptors mixed with a test substance, and a control group may be measured, and the respective measurement results may be compared. By comparing the responses of the test group with the responses of the control group, for example, the effect of the test substance on the olfactory receptors in suppressing the aldehyde odor can be evaluated.

[0035] 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 not contacted with a lower aldehyde; 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.

[0036] In one embodiment, for example, if the response in a test group using the test substance is suppressed compared to the control group, the test substance can be selected as an inhibitor of an aldehyde odor. 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 an aldehyde odor.

[0037] For example, if the response of the olfactory receptor to the test substance does not change in the reference data as the amount of the test substance used increases, and the response of the olfactory receptor to the lower aldehyde decreases in the test data after contact with the lower aldehyde, the test substance mixed with the specific olfactory receptor functions as an antagonist. In this case, the test substance is considered to be useful as an agent for suppressing aldehyde odors. In this way, after mixing the olfactory receptor with the test substance, the test substance that inhibits the contact response of the olfactory receptor to the lower aldehyde can be selected as an inhibitor of the aldehyde odor.

[0038] The specific method for measuring the olfactory receptor response is not particularly limited as long as it allows for evaluation of the olfactory receptor response. For example, measurement of the amount of intracellular cAMP can be used. The olfactory receptor response can be evaluated by using the amount of intracellular cAMP as an indicator of the olfactory receptor response. 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.

[0039] In one embodiment, OR6B1 can be replaced by a polypeptide having a function equivalent to OR6B1. The amino acid sequence of the polypeptide having a function equivalent to OR6B1 preferably exhibits 80% or more homology with the amino acid sequence of OR6B1, 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.

[0040] In one embodiment, Olfr449 can be replaced by a polypeptide having a function equivalent to Olfr449. The amino acid sequence of the polypeptide having a function equivalent to Olfr449 preferably exhibits 80% or more homology with the amino acid sequence of Olfr449, 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.

[0041] In addition to OR6B1 and Olfr449, any polypeptide with equivalent function can be used as an olfactory receptor. For example, homologous olfactory receptors from animals other than humans and mice can be used. Examples of animals other than humans and mice include rats and other experimental model organisms.

[0042] The olfactory receptor can be used in any manner as long as it does not lose its responsiveness to lower aldehydes.For example, the olfactory receptor can be used in the following manner: 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.In addition, tissues having olfactory mucus (such as the olfactory epithelium and the olfactory mucosa) can be used as the olfactory receptor.

[0043] 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, it is preferable to use human-derived genetically modified cells that express an olfactory receptor. 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 expression of olfactory receptors 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, 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.

[0044] 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 lower aldehyde is supplied around the cell culture plate to bring the lower aldehyde 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 a lower aldehyde so that the response start time 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.

[0045] In one embodiment, a sensory test may be further performed. For example, a test substance that is expected to be useful as an aldehyde odor suppressant may be selected as a candidate substance for an aldehyde odor suppressant or a deodorizer, and the aldehyde odor suppression effect of the candidate substance may be evaluated by a sensory test. A candidate substance that is confirmed to have an aldehyde odor suppression effect in the sensory test may be selected as an aldehyde odor suppressant or a deodorizer.

[0046] The sensory test can be performed in accordance with a typical procedure for evaluating deodorants. For example, the evaluator may smell the aldehyde odor simultaneously with the odor of the candidate substance and evaluate the intensity of the aldehyde odor, or may smell the aldehyde odor separately from the odor of the candidate substance and evaluate the intensity of the aldehyde odor. The evaluation results obtained are compared with the intensity of the aldehyde odor alone. Candidate substances that are evaluated as reducing the intensity of the aldehyde odor as a result of the sensory test are expected to be useful as aldehyde odor suppressants.

[0047] (Application) In one embodiment of the screening method, an inhibitor of aldehyde odor is obtained. The obtained inhibitor of aldehyde odor can be used as an active ingredient of a deodorizer for aldehyde odor. The specific manner of use of the deodorant is not particularly limited. For example, the agent for suppressing aldehyde odor may be used as an active ingredient in a composition or article for suppressing aldehyde odor, or may be used in the production of a composition or article for suppressing aldehyde odor. The searching method according to one embodiment can also be suitably applied to the use in a method for producing a deodorant.

[0048] Examples of uses for deodorizers include human and animal toilet or excrement treatment; excrement treatment in medical facilities, nursing homes, and care facilities; disposable diapers, sanitary products; odor treatment inside automobiles, exhaust fumes, smoking spaces, and care spaces, particularly odor treatment targeting breath, body odor, and bodily fluids after drinking; clothing accessories 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.; treatment of lower aldehydes derived from building materials; and manufacturing equipment for products that emit aldehyde odors. However, the applications of aldehyde odor suppressants are not limited to these examples.

[0049] (Action and effect) In the screening method according to one embodiment described above, a specific olfactory receptor is mixed with a test substance. Because the specific olfactory receptor specifically responds to aldehyde odors, it is possible to biochemically test whether the test substance functions as an inhibitor of aldehyde odors. Therefore, it is possible to obtain an inhibitor that is likely to fully exert its deodorizing effect against aldehyde odors.

[0050] In addition, aldehyde odor inhibitors suppress the response of specific olfactory receptors to aldehyde odors. Therefore, the concentration required to achieve the same level of deodorizing effect in a target space can likely be set lower than in the case of conventional physical or chemical deodorizers. Therefore, aldehyde odor inhibitors are practical as active ingredients in aldehyde odor deodorizers.

[0051] According to one embodiment of the screening method, an aldehyde odor suppressant, i.e., an olfactory receptor antagonist, can be used as an active ingredient in a deodorant. Because the antagonist antagonizes the aldehyde odor, inhibiting the olfactory receptor response and the perception of malodor, there is no need to maintain a pre-dispersed state, as is the case with conventional physical or chemical deodorizers. Therefore, antagonist aldehyde odor suppressants are also suitable for instant deodorization applications, such as sprays. [Example]

[0052] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0053] <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 MluI 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.

[0054] [Table 1]

[0055] [Table 2]

[0056] 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, producing Hana3A cells expressing each of the 392 human olfactory receptors listed in Tables 1 and 2.

[0057] [Table 3]

[0058] <Lower aldehydes> The following lower aldehydes were used as odorants: Acetaldehyde Propanal Butanal Pentanal

[0059] <Aldehydes with 6 or more carbon atoms> The following aldehydes with six or more carbon atoms were used as odorants. Hexanal Heptanal Octanal Nonanal Decanal Undecanal Dodecanal

[0060] <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, luciferase-derived luminescence was measured 10 times every minute starting immediately after stimulation, and the area under the curve obtained was used as the response value. Similar measurements were also performed under unstimulated conditions, and the response intensity was calculated by subtracting the luminescence value under stimulated conditions from the luminescence value under unstimulated conditions.

[0061] <Search for olfactory receptors that respond to lower aldehydes> (Identification of OR6B1) 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 the cAMP Reagent to be introduced into the cells. Finally, an aqueous solution of acetaldehyde was placed on the top of the wall between the wells of the 96-well plate, and the lid of the well plate was closed. The acetaldehyde was allowed to volatilize on the inside of the lid, bringing the olfactory receptor-expressing cells into contact with the odorant. The Glo Sensor assay was then performed to measure the response strength (fold increase) of the olfactory receptor to malodorous molecules. The results are shown in Figure 1.

[0062] The vertical axis in Figure 1 shows the relative response strength of each receptor-expressing cell to acetaldehyde (Figure 1). The relative response strength was calculated assuming that the response strength in the absence of acetaldehyde was 1. The response strength under the same concentration of acetaldehyde and the same exposure time was also set to 1. The response to acetaldehyde was measured for each cell type expressing 392 types of olfactory receptors.

[0063] As a result, OR6B1 was identified as the olfactory receptor that showed the highest response to acetaldehyde (Figure 1). Furthermore, although not shown in the figure, the results of a separate experiment conducted by the present inventors revealed that Olfr449, a mouse homologue of OR6B1, also responds strongly to acetaldehyde.

[0064] (Concentration dependence of OR6B1 response to lower aldehydes) The response strength of OR6B1 to different concentrations of acetaldehyde was measured. The results are shown in Figure 2. As a result, OR6B1 showed a concentration-dependent response to acetaldehyde, confirming that it is an acetaldehyde receptor.

[0065] (Confirmation of OR6B1 response to lower aldehydes) The response of OR6B1 was confirmed using acetaldehyde, propanal, butanal, and pentanal as lower aldehydes. The response of OR6B1 was confirmed using hexanal, heptanal, octanal, nonanal, decanal, undecanal, and dodecanal as aldehydes with six or more carbon atoms. The lower aldehydes and aldehydes with six or more carbon atoms were all brought into contact with OR6B1 in a gaseous state, and the response intensity of OR6B1 was then measured.

[0066] To measure the response intensity, OR6B1-expressing cells were stimulated by contacting them with OR6B1 so that the final concentration of each reagent was 100 μM. Luminescence values ​​were measured over time 19 times at 1-minute intervals using a GloMax Discover Microplate Reader (Promega). The response intensity was calculated from the total area value of the 19 measurements. The results are shown in Figure 3. The "Control" in Figure 3 shows the luminescence value of OR6B1 in the absence of stimulation by lower aldehydes or aldehydes with six or more carbon atoms. The relative intensity to this control luminescence value was calculated and is shown in Figure 3. These results confirmed that OR6B1 responds to these lower aldehydes, but does not respond to aldehydes with six or more carbon atoms, making it a receptor that specifically responds to lower aldehydes.

[0067] <Test substance> 105 compounds as test substances were diluted with HBSS buffer containing 10 mM HEPES to a final concentration of 100 μM.

[0068] <Search for Antagonists of OR6B1> The medium was removed from the cultures of OR6B1-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. Next, the test substance was added to each well of the 96-well plate to a final concentration of each concentration shown in Table 4, and a GloSensor assay was performed 10 minutes later to measure the response intensity (fold increase) of the olfactory receptor to the test substance and obtain reference data. Thereafter, a 1% aqueous solution of acetaldehyde as an odorant molecule was placed on the upper surface of the wall portion forming the walls between the wells of a 96-well plate, and then the lid of the well plate was closed. By volatilizing acetaldehyde in the well plate, the olfactory receptor-expressing cells were brought into contact with the odorant molecule. In this state, a GloSensor assay was performed to measure the response intensity (fold increase) of the olfactory receptor to the odorant molecule and obtain test data. The measurement results of the response intensity are shown in Table 4.

[0069]

Table 4

[0070] As shown in Table 4, the response intensity measurements identified the following antagonists: ethyl salicylate, ethylene brassylate, fractone, acetylcedrene, α-damascone, β-ionone, citronellolnitrile, D-limonene, and methyl benzoate. These nine compounds suppressed the OR6B1 response to acetaldehyde, suggesting that they function as OR6B1 antagonists. Furthermore, these nine compounds are thought to be inhibitors that are likely to exert a sufficient deodorizing effect against aldehyde odors. On the other hand, estragole, phenylacetaldehyde dimethyl acetal, and hexyl salicylate did not show any inhibitory effect on OR6B1's response to acetaldehyde. [Industrial Applicability]

[0071] According to the present invention, it is possible to obtain an inhibitor that is likely to exhibit a sufficient deodorizing effect against aldehyde odors.

Claims

1. A method for searching for an agent for suppressing aldehyde odor, comprising: mixing a test substance with at least one or more olfactory receptors selected from the group consisting of OR6B1, Olfr449, an olfactory receptor showing 90% or more homology to the amino acid sequence of OR6B1, and an olfactory receptor showing 90% or more homology to the amino acid sequence of Olfr449; The olfactory receptor responds to lower aldehydes having 1 to 4 carbon atoms, and the method for searching for an inhibitor of aldehyde odor is also provided.

2. The screening method according to claim 1, further comprising contacting the olfactory receptor with the lower aldehyde having 1 to 4 carbon atoms after mixing the olfactory receptor with the test substance.

3. The screening method according to claim 2, further comprising contacting the olfactory receptor with the lower aldehyde having 1 to 4 carbon atoms, and then selecting a test substance that inhibits the response of the olfactory receptor as an inhibitor of aldehyde odor.

4. The screening method according to any one of claims 1 to 3, wherein the lower aldehyde is at least one selected from the group consisting of acetaldehyde, propanal, and butanal.

5. The screening method according to any one of claims 1 to 4, wherein the inhibitor is an antagonist of the olfactory receptor.

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