JP2015202075A - Search method of butyl acrylate odor inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify substances inhibiting butyl acrylate odor.SOLUTION: A search method of butyl acrylate odor inhibitor comprises the steps of: adding test materials and butyl acrylate into at least one kind of olfactory receptor polypeptides selected from a group consisting of OR1A1, OR2W1, OR4Q3, and polypeptides having at least 80% amino acid sequence identity to each of them; measuring a response of the olfactory receptor polypeptide to butyl acrylate; and identifying the test materials inhibiting the response of the olfactory receptor polypeptide on the basis of the response measured.

Description

  The present invention relates to a method for searching for a butyl acrylate odor inhibitor.

  Butyl acrylate (BA) is an ester of acrylic acid and normal butanol, and is used as a raw material for chemical products such as fiber treatment agents, adhesives, paints, synthetic resins, acrylic rubbers, and emulsions. However, since butyl acrylate has a strong ester odor, an unpleasant odor may be generated in the manufacturing process of the product, or the product itself may emit an unpleasant odor.

  In the development of conventional fragrance substances and deodorant substances, odor evaluation has been carried out by sensory tests by experts. However, the sensory test has problems such as the need to train experts who can evaluate odors and low throughput.

  In mammals such as humans, the smell is bound to the olfactory receptor on the olfactory nerve cell located in the olfactory epithelium in the upper nasal cavity, and the response of the receptor is transmitted to the central nervous system. It is recognized by. In the case of humans, it is reported that there are about 400 olfactory receptors, and the genes encoding them are about 2% of all human genes. In general, olfactory receptors and odor molecules are associated with each other in a plurality of combinations. That is, each olfactory receptor receives a plurality of odor molecules with similar structures with different affinities, while each odorant receptor is received by a plurality of olfactory receptors. Furthermore, it has been reported that an odor molecule that activates one olfactory receptor acts as an antagonist that inhibits activation of another olfactory receptor. The combination of these multiple olfactory receptor responses has led to the recognition of individual odors.

  Therefore, even when the same odor molecule is present, if another odor molecule is present at the same time, the receptor response is inhibited by the other odor molecule, and the finally recognized odor may be completely different. This mechanism is called olfactory receptor antagonism. Unlike the deodorization method by adding another odor such as perfume or fragrance, the modulation of odor by this receptor antagonism can specifically lose recognition of specific bad odors. Since there is no discomfort due to the smell of the agent, this is a preferred deodorizing means. Patent Documents 1 and 2 describe that substances that suppress malodor such as hexanoic acid and skatole can be searched by olfactory receptor antagonism using olfactory receptor activity as an index. Patent Document 3 also describes searching for a substance that suppresses sweat odor by using the activity of an olfactory receptor as an index. Patent Document 4 describes a method for identifying a drug that modulates the function of a polypeptide by measuring the activity of the polypeptide encoding an olfactory receptor in the presence of isovaleric acid or its equivalent. Yes. Patent Document 5 describes a method of screening a library of chemical substances for compounds related to olfactory sensation by identifying a compound that specifically binds to a polypeptide encoding an olfactory receptor.

JP 2012-050411 A JP 2012-249614 A US Patent Publication No. 2013/0336910 International Publication No. 2006/094704 JP-T-2004-504010

  The present invention provides a method for efficiently searching for a butyl acrylate odor suppressor using the response of an olfactory receptor as an index.

  The present inventors succeeded in newly identifying an olfactory receptor that responds to butyl acrylate. In addition, the present inventor can evaluate and select a substance that suppresses butyl acrylate odor by masking with olfactory receptor antagonism by using the response of the olfactory receptor or a polypeptide having the same function as an index. I found out.

Accordingly, the present invention is a method for searching for a butyl acrylate odor suppressor comprising the following:
Adding a test substance and butyl acrylate to at least one olfactory receptor polypeptide selected from the group consisting of OR1A1, OR2W1, OR4Q3 and a polypeptide having at least 80% identity in amino acid sequence with OR1A1, OR2W1, OR4Q3 ;
Measuring the response of the olfactory receptor polypeptide to butyl acrylate; and
Identifying a test substance that suppresses the response of the olfactory receptor polypeptide based on the measured response;
A method comprising:

  ADVANTAGE OF THE INVENTION According to this invention, the butyl acrylate odor inhibitor which can selectively deodorize butyl acrylate odor by masking by an olfactory receptor antagonism can be searched efficiently.

Response of olfactory receptors to butyl acrylate. The horizontal axis represents individual olfactory receptors, and the vertical axis represents response intensity (n = 2). Response of olfactory receptors to various concentrations of butyl acrylate. A: OR1A1, B: OR2W1, C: OR4Q3. n = 3-4, error bar = ± SE.

  In this specification, the term “masking” relating to odor refers to all means for making the target odor unrecognized or weakening recognition. “Masking” may include chemical means, physical means, biological means, and sensory means. For example, as masking, any means for removing odor molecules that cause the target odor from the environment (for example, adsorption and chemical decomposition of odor molecules), to prevent the target odor from being released into the environment. (For example, containment), a method of adding another odor such as a fragrance or a fragrance to make it difficult to recognize the target odor, and the like.

  In the present specification, “masking by olfactory receptor antagonism” is one form of the above-mentioned “masking” in a broad sense, and by applying the target odor molecule and other odor molecules together, It is a means of inhibiting the receptor response to a target odor molecule by a molecule and consequently changing the odor recognized by an individual. Masking by olfactory receptor antagonism is distinguished from means for canceling the target odor by another strong odor, such as a fragrance, even if the means uses other odor molecules. An example of masking by olfactory receptor antagonism is the case of using a substance that inhibits the response of the olfactory receptor, such as an antagonist (antagonist). If a substance that inhibits the response is applied to the receptor of the odor molecule that causes a specific odor, the response of the receptor to the odor molecule is suppressed, so that the odor finally perceived by the individual is changed. Can do.

  In the present specification, “olfactory receptor polypeptide” refers to an olfactory receptor or a polypeptide having a function equivalent thereto, and a polypeptide having a function equivalent to an olfactory receptor is similar to an olfactory receptor. A function that increases the amount of intracellular cAMP by activating adenylate cyclase in combination with intracellular Gαs, which can be expressed on the cell membrane and activated by binding of odor molecules (Nat. Neurosci., 2004, 5: 263-278).

  In the present specification, nucleotide sequence and amino acid sequence identity is calculated by the Lippman-Pearson method (Lipman-Pearson method; Science, 1985, 227: 1435-41). Specifically, using the homology analysis (Search homology) program of genetic information software Genetyx-Win (Ver. 5.1.1; software development), analysis is performed with Unit size to compare (ktup) as 2. Is calculated by

  As shown in FIG. 1, the present inventor has identified OR1A1, OR2W1, and OR4Q3 as olfactory receptors that specifically respond to butyl acrylate among many olfactory receptors. Furthermore, as shown in FIG. 2, OR1A1, OR2W1, and OR4Q3 all respond to butyl acrylate in a concentration-dependent manner. Therefore, a substance that suppresses the response of OR1A1, OR2W1 or OR4Q3, or a polypeptide having a function similar to these causes a change in the recognition of butyl acrylate odor in the center by masking based on olfactory receptor antagonism, As a result, the butyl acrylate odor can be selectively suppressed.

  Accordingly, the present invention provides a method for searching for a butyl acrylate odor suppressor. The method involves the addition of a test substance and butyl acrylate to at least one olfactory receptor polypeptide selected from the group consisting of OR1A1, OR2W1, OR4Q3, and a polypeptide having at least 80% identity in amino acid sequence thereto. Measuring the response of the olfactory receptor polypeptide to butyl acrylate; and identifying a test substance that suppresses the response of the olfactory receptor polypeptide based on the measured response. Including. The identified test substance is selected as a butyl acrylate odor suppressor. The method of the present invention may be a method performed in vitro or ex vivo.

  In the method of the present invention, a test substance and butyl acrylate are added to an olfactory receptor polypeptide that is responsive to butyl acrylate. Butyl acrylate can be purchased from, for example, Mitsubishi Chemical Corporation.

  The test substance used in the method of the present invention is not particularly limited as long as it is a substance desired to be used as a butyl acrylate odor suppressor. The test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, etc., or may be a compound, a composition or a mixture. Good.

  The olfactory receptor polypeptide used in the method of the present invention is at least one selected from the group consisting of OR1A1, OR2W1, OR4Q3, and polypeptides having at least 80% identity in amino acid sequence.

  OR1A1, OR2W1, and OR4Q3 are all olfactory receptors expressed in human olfactory cells. OR1A1 is registered in GenBank as GI: 289547622. OR1A1 is a protein consisting of an amino acid sequence represented by SEQ ID NO: 2 encoded by a gene having a nucleotide sequence represented by SEQ ID NO: 1. OR2W1 is registered with GenBank as GI: 16234788. OR2W1 is a protein consisting of an amino acid sequence represented by SEQ ID NO: 4 encoded by a gene having a nucleotide sequence represented by SEQ ID NO: 3. OR4Q3 is registered in GenBank as GI: 53828745. OR4Q3 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 6 encoded by the gene having the nucleotide sequence represented by SEQ ID NO: 5.

  The polypeptide having at least 80% identity in the amino acid sequence with OR1A1 is at least 80%, for example, 80% or more, preferably 85% or more, more preferably 90% or more, further with the amino acid sequence represented by SEQ ID NO: 2. Preferred is a polypeptide having an amino acid sequence having an identity of 95% or more, still more preferably 98% or more, and still more preferably 99% or more, and responsiveness to butyl acrylate.

  The polypeptide having at least 80% identity in the amino acid sequence with OR2W1 includes at least 80%, for example, 80% or more, preferably 85% or more, more preferably 90% or more, more than the amino acid sequence represented by SEQ ID NO: 4. Preferred is a polypeptide comprising an amino acid sequence having an identity of 95% or more, still more preferably 98% or more, and still more preferably 99% or more, and responsive to butyl acrylate.

  The polypeptide having at least 80% identity in the amino acid sequence with OR4Q3 includes at least 80%, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and the amino acid sequence represented by SEQ ID NO: 6. Preferred is a polypeptide having an amino acid sequence having an identity of 95% or more, still more preferably 98% or more, and still more preferably 99% or more, and responsiveness to butyl acrylate.

  In the method of the present invention, at least one selected from the above-described olfactory receptor polypeptides may be used, but any two or more may be used in combination. Preferably, OR1A1 or a polypeptide having at least 80% identity in amino acid sequence with OR1A1, a polypeptide having at least 80% identity in amino acid sequence with OR2W1, and at least 80% in amino acid sequence with OR4Q3 or this At least one selected from the group consisting of polypeptides having the same identity is used, and more preferably, OR1A1 or a polypeptide having at least 80% identity in amino acid sequence, OR2W1 or in amino acid sequence with this At least two selected from the group consisting of polypeptides having at least 80% identity and OR4Q3 or polypeptides having at least 80% identity in amino acid sequence are used, more preferably, R1A1 or polypeptide having at least 80% identity in amino acid sequence, OR2W1 or polypeptide having at least 80% identity in amino acid sequence, and OR4Q3 or at least 80% identity in amino acid sequence Are used in combination, more preferably OR1A1, OR2W1 and OR4Q3 are used in combination.

  In the method of the present invention, the olfactory receptor polypeptide can be used in any form as long as it does not lose responsiveness to butyl acrylate. For example, the olfactory receptor polypeptide is a tissue or cell that naturally expresses the olfactory receptor polypeptide, such as an olfactory receptor or olfactory cell isolated from a living body, or a culture thereof; the olfactory receptor A membrane of an olfactory cell carrying a polypeptide; a recombinant cell or culture thereof genetically engineered to express the olfactory receptor polypeptide; a membrane of the recombinant cell having the olfactory receptor polypeptide; It can be used in the form of an artificial lipid bilayer membrane having the olfactory receptor polypeptide. All of these forms fall within the scope of the olfactory receptor polypeptide used in the present invention.

  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 genetically engineered to express the olfactory receptor polypeptide. Cells or cultures thereof are used. The recombinant cell can be produced by transforming the cell using a vector incorporating a gene encoding the olfactory receptor polypeptide.

  Preferably, in order to promote cell membrane expression of the olfactory receptor polypeptide, a gene encoding 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 a cell together with a gene encoding the olfactory receptor polypeptide. An example of RTP1S includes human RTP1S. Human RTP1S is a protein consisting of the amino acid sequence represented by SEQ ID NO: 8, which is registered in GenBank as GI: 50234917 and is encoded by a gene having a nucleotide sequence represented by SEQ ID NO: 7.

  According to the method of the present invention, following the addition of the test substance and butyl acrylate to the olfactory receptor polypeptide, the response of the olfactory receptor polypeptide to butyl acrylate is measured. The measurement may be performed by any method known in the art as a method for measuring the response of the olfactory receptor, for example, measurement of intracellular cAMP amount. For example, when the olfactory receptor is activated by an odor molecule, it is known to increase the amount of intracellular cAMP by activating adenylate cyclase in combination with intracellular Gαs (Nat. Neurosci., 2004, 5: 263-278). Therefore, the response of the olfactory receptor polypeptide can be measured by using the amount of intracellular cAMP after addition of the odor molecule as an index. Examples of the method for measuring the amount of cAMP include an ELISA method and a reporter gene assay. Other methods for measuring the response of the olfactory receptor polypeptide include calcium imaging.

  Next, based on the measured response of the olfactory receptor polypeptide, the effect of the test substance on the response to butyl acrylate is evaluated, and the test substance that suppresses the response is identified. The effect of the test substance can be evaluated by comparing the response of the olfactory receptor polypeptide to which the test substance is added to butyl acrylate to the response to butyl acrylate in the control group. As a control group, the olfactory receptor polypeptide to which the test substance of different concentration was added, the olfactory receptor polypeptide to which the test substance was not added, the olfactory receptor polypeptide to which the control substance was added, and the test substance were added. Examples of the olfactory receptor polypeptide may be mentioned.

  For example, the effect of the test substance on the response of the olfactory receptor polypeptide is between the test substance addition group of the higher concentration and the test substance addition group of the lower concentration, the test substance addition group and the non-addition group. The olfactory receptor polypeptide can be evaluated by comparing the response of the olfactory receptor polypeptide to butyl acrylate between the test substance addition group and the control substance addition group, or before and after the test substance addition. A substance that suppresses the response of the olfactory receptor polypeptide to butyl acrylate when the response of the olfactory receptor polypeptide is suppressed by addition of the test substance or by addition of a higher concentration of the test substance Can be identified as

  For example, the response of the olfactory receptor polypeptide in the test substance addition group measured by the above procedure is preferably suppressed to 60% or less, more preferably 50% or less, and even more preferably 25% or less compared to the control group. If so, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to butyl acrylate. Alternatively, if the response of the olfactory receptor polypeptide in the test substance addition group measured by the above procedure is statistically significantly suppressed as compared with the control group, the test substance is added to butyl acrylate. It can be identified as a substance that suppresses the response of the olfactory receptor polypeptide.

  The test substance identified by the above procedure is a substance that can suppress the recognition of the odor of butyl acrylate by an individual by suppressing the response of the olfactory receptor to butyl acrylate. Therefore, the test substance identified by the above procedure can be selected as a butyl acrylate odor suppressor. The substance selected as the butyl acrylate odor suppressor by the method of the present invention can suppress the butyl acrylate odor by olfactory masking based on the suppression of the response of the olfactory receptor to butyl acrylate.

  Accordingly, in one embodiment, the material selected by the method of the present invention may be an active ingredient of a butyl acrylate odor suppressor. Or the substance selected by the method of this invention can be contained in the compound or composition for suppressing a butyl acrylate odor as an active ingredient for suppressing a butyl acrylate odor. Alternatively, the substance selected by the method of the present invention can be used for the production of a butyl acrylate odor inhibitor or for the production of a compound or composition for inhibiting butyl acrylate odor. .

  As exemplary embodiments of the present invention, the following compositions, production methods, uses or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

<1> A search method for a butyl acrylate odor inhibitor, which is
Adding a test substance and butyl acrylate to at least one olfactory receptor polypeptide selected from the group consisting of OR1A1, OR2W1, OR4Q3 and a polypeptide having at least 80% identity in amino acid sequence with OR1A1, OR2W1, OR4Q3 ;
Measuring the response of the olfactory receptor polypeptide to butyl acrylate; and
Identifying a test substance that suppresses the response of the olfactory receptor polypeptide based on the measured response;
Including methods.

<2> The method according to <1> above, wherein preferably, the OR1A1 is a protein comprising the amino acid sequence represented by SEQ ID NO: 2, and the OR2W1 is a protein comprising the amino acid sequence represented by SEQ ID NO: 4 And the OR4Q3 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 6.

<3> The method according to <1> or <2> above,
A polypeptide having at least 80% identity in amino acid sequence with the OR1A1 is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, even more, with the amino acid sequence represented by SEQ ID NO: 2. Preferably, it is a polypeptide consisting of an amino acid sequence having an identity of 95% or more, more preferably 98% or more, even more preferably 99% or more, and preferably responsive to butyl acrylate,
The polypeptide having at least 80% identity in the amino acid sequence with OR2W1 is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, even more, with the amino acid sequence represented by SEQ ID NO: 4. Preferably, it is a polypeptide consisting of an amino acid sequence having an identity of 95% or more, more preferably 98% or more, even more preferably 99% or more, and preferably responsive to butyl acrylate,
And the polypeptide having at least 80% identity in amino acid sequence with OR4Q3 is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, further with the amino acid sequence represented by SEQ ID NO: 6 More preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more of an amino acid sequence, and preferably a polypeptide responsive to butyl acrylate,
Method.

<4> The method according to any one of <1> to <3> above, from the group consisting of the OR1A1, OR2W1, OR4Q3, and a polypeptide having at least 80% identity in amino acid sequence with these. At least one olfactory receptor polypeptide selected is
Preferably, a polypeptide having at least 80% identity in amino acid sequence with OR1A1, or a polypeptide having at least 80% identity in amino acid sequence with OR2W1, and at least 80% identity in amino acid sequence with OR4Q3 At least one selected from the group consisting of polypeptides having
More preferably, a polypeptide having at least 80% identity in amino acid sequence with OR1A1, or a polypeptide having at least 80% identity in amino acid sequence with OR2W1, and at least 80% identity in amino acid sequence with OR4Q3 At least two selected from the group consisting of polypeptides having sex;
More preferably, a polypeptide having at least 80% identity in amino acid sequence with OR1A1, or a polypeptide having at least 80% identity in amino acid sequence with OR2W1, and at least 80% identity in amino acid sequence with OR4Q3 A combination of polypeptides having sex,
Preferably, it is a combination of OR1A1, OR2W1, and OR4Q3.
Method.

<5> The method according to any one of <1> to <4> above,
Preferably, the method further comprises measuring a response to butyl acrylate in the control group, and the control group comprises:
The olfactory receptor polypeptide to which the test substance has not been added;
The olfactory receptor polypeptide to which a control substance is added; or the olfactory receptor polypeptide before the addition of the test substance.

<6> The method according to <5> above, preferably
When the response of the olfactory receptor polypeptide to which the test substance is added to butyl acrylate is statistically significantly suppressed as compared to the response to butyl acrylate in the control group, the test substance is Identifying as a substance that suppresses the response of the olfactory receptor polypeptide to butyl acid,
Further comprising a method.

<7> The method according to <5> above, preferably
The response of the olfactory receptor polypeptide to which the test substance is added to butyl acrylate is preferably 60% or less, more preferably 50% or less, and even more preferably 25% of the response to butyl acrylate in the control group. Identifying the test substance as a substance that suppresses the response of the olfactory receptor polypeptide to butyl acrylate, if suppressed to:
Further comprising a method.

<8> The method according to any one of <1> to <7>, wherein a test substance that suppresses a response of the olfactory receptor polypeptide is preferably selected as a butyl acrylate odor suppressor. Further comprising a method.

<9> The method according to any one of <1> to <8> above,
Preferably, at least one olfactory receptor polypeptide selected from the group consisting of the OR1A1, OR2W1, OR4Q3, and a polypeptide having at least 80% identity in amino acid sequence with the OR1A1, OR2W1, OR4Q3, Expressed on recombinant cells genetically engineered to express
Method.

<10> The method according to <9> above, wherein the recombinant cell is
Preferably, a cell into which a gene encoding the olfactory receptor polypeptide and a gene encoding RTP1S are introduced,
More preferably, a cell into which a gene encoding the olfactory receptor polypeptide and a gene encoding human RTP1S consisting of the amino acid sequence represented by SEQ ID NO: 8 have been introduced.
Method.

<11> The method according to any one of <1> to <8> above,
Preferably, the at least one olfactory receptor polypeptide selected from the group consisting of OR1A1, OR2W1, OR4Q3, and a polypeptide having at least 80% identity in amino acid sequence with the above <9> or <9>10> or a recombinant cell thereof is used,
Method.

<12> The method according to any one of <1> to <11> above,
Preferably, the measurement of the response of the olfactory receptor polypeptide is measurement of intracellular cAMP amount by ELISA or reporter gene assay, or measurement by calcium imaging.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Example 1 Identification of Olfactory Receptor Responsive to Butyl Acrylate 1) Cloning of Human Olfactory Receptor Gene Human olfactory receptor is a human genomic DNA female (G1521: Promega) based on sequence information registered in GenBank. Cloning was performed by PCR using a template. Each gene amplified by the PCR method is incorporated into a pENTR vector (Invitrogen) according to the manual, and NotI and AscI prepared downstream of the Flag-Rho tag sequence on the pME18S vector using the NotI and AscI sites present on the pENTR vector. Recomposed to the site.

2) Preparation of pME18S-human RTP1S vector The RTP1S gene (SEQ ID NO: 7) encoding RTP1S (SEQ ID NO: 8) was incorporated into the EcoRI and XhoI sites of the pME18S vector.

3) Preparation of olfactory receptor-expressing cells HEK293 cells each expressing 417 types of human olfactory receptors were prepared. A reaction solution having the composition shown in Table 1 was prepared and allowed to stand in a clean bench for 15 minutes, and then added to each well of a 96-well plate (BD). Next, 100 μL of HEK293 cells (3 × 10 ⁵ cells / cm ² ) were seeded in each well and cultured in an incubator maintained at 37 ° C. and 5% CO ₂ for 24 hours.

4) Luciferase assay The olfactory receptor expressed in HEK293 cells increases the amount of intracellular cAMP by coupling with intracellular Gαs and activating adenylate cyclase. For the measurement of odor response in this study, a luciferase reporter gene assay was used which monitors the increase in intracellular cAMP level as a luminescence value derived from the firefly luciferase gene (fluc2P-CRE-hygro). In addition, a renilla luciferase gene fused to the downstream of the CMV promoter (hRluc-CMV) was introduced at the same time, and used as an internal standard for correcting errors in gene transfer efficiency and cell number.
The medium was removed from the culture prepared in 3) above, and 75 μL of a solution containing an odorant (butyl acrylate 300 μM) prepared with CD293 medium (Invitrogen) was added. The cells were cultured for 2.5 hours in a CO ₂ incubator to fully express the luciferase gene in the cells. The luciferase activity was measured using a Dual-Glo ^™ luciferase assay system (Promega) according to the operation manual of the product. A value obtained by dividing the luminescence value derived from firefly luciferase induced by odor substance stimulation by the luminescence value in cells not subjected to odor substance stimulation was calculated as a fold increase, and used as an index of response intensity.

5) Results As a result of measuring responses to butyl acrylate 300 μM for 417 types of olfactory receptors, OR1A1, OR2W1, and OR4Q3 showed responses (FIG. 1). The response of OR1A1, OR2W1 and OR4Q3 to butyl acrylate was concentration dependent (FIG. 2). OR1A1, OR2W1 and OR4Q3 are novel butyl acrylate receptors that have not previously been found to respond to butyl acrylate.

Claims (7)

A method for searching for a butyl acrylate odor suppressor comprising:
Adding a test substance and butyl acrylate to at least one olfactory receptor polypeptide selected from the group consisting of OR1A1, OR2W1, OR4Q3 and a polypeptide having at least 80% identity in amino acid sequence with OR1A1, OR2W1, OR4Q3 ;
Measuring the response of the olfactory receptor polypeptide to butyl acrylate; and
Identifying a test substance that suppresses the response of the olfactory receptor polypeptide based on the measured response;
Including methods.   The OR1A1 is a protein comprising the amino acid sequence represented by SEQ ID NO: 2, the OR2W1 is a protein comprising the amino acid sequence represented by SEQ ID NO: 4, and the OR4Q3 is a protein comprising the amino acid sequence represented by SEQ ID NO: 6. The method of claim 1, wherein: The method according to claim 1 or 2, wherein
A polypeptide having at least 80% identity in the amino acid sequence with OR1A1 comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2, and is responsive to butyl acrylate A peptide,
A polypeptide having at least 80% identity in amino acid sequence with OR2W1 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 4, and is responsive to butyl acrylate A peptide,
The polypeptide having at least 80% identity in amino acid sequence with OR4Q3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 6, and has responsiveness to butyl acrylate A polypeptide,
Method.   4. The method of any one of claims 1-3, wherein the olfactory receptor polypeptide is expressed on a recombinant cell that has been genetically engineered to express the olfactory receptor polypeptide.   The method according to any one of claims 1 to 4, further comprising measuring a response of the olfactory receptor polypeptide to which no test substance has been added to butyl acrylate.   The response of the olfactory receptor polypeptide to which the test substance is added to butyl acrylate is suppressed to 60% or less of the response of the olfactory receptor polypeptide to which the test substance is not added to butyl acrylate. 6. The method of claim 5, further comprising identifying the test substance as a substance that suppresses the response of the olfactory receptor polypeptide to butyl acrylate.   The method according to any one of claims 1 to 6, wherein the measurement of the response of the olfactory receptor polypeptide is measurement of intracellular cAMP amount by ELISA or reporter gene assay, or measurement by calcium imaging.