WO2016204211A1 - Method for evaluating and selecting agent for suppressing odors of sulfide compounds - Google Patents

Method for evaluating and selecting agent for suppressing odors of sulfide compounds Download PDF

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WO2016204211A1
WO2016204211A1 PCT/JP2016/067894 JP2016067894W WO2016204211A1 WO 2016204211 A1 WO2016204211 A1 WO 2016204211A1 JP 2016067894 W JP2016067894 W JP 2016067894W WO 2016204211 A1 WO2016204211 A1 WO 2016204211A1
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olfactory receptor
response
receptor polypeptide
sulfide compound
test substance
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PCT/JP2016/067894
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French (fr)
Japanese (ja)
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敬一 吉川
菜穂子 齋藤
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花王株式会社
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Priority claimed from JP2016116227A external-priority patent/JP6122181B2/en
Application filed by 花王株式会社 filed Critical 花王株式会社
Priority to EP16811685.3A priority Critical patent/EP3312289B1/en
Priority to US15/737,079 priority patent/US10585087B2/en
Publication of WO2016204211A1 publication Critical patent/WO2016204211A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for evaluating or selecting an inhibitor for odor of sulfide compounds.
  • Volatile sulfur compounds generated from wastes such as drain outlets and garbage are malodorous components that make people uncomfortable even at low concentrations. These volatile sulfur compounds are produced by degradation of sulfur-containing amino acids such as cysteine and methionine contained in sewage and wastes or proteins containing them by the action of metabolic enzymes such as methionine lyase and cysteine lyase in bacteria. appear. For example, among volatile sulfur compounds that cause unpleasant odor, methyl mercaptan is produced from methionine by the action of methionine lyase, and hydrogen sulfide is produced from cysteine by the action of cysteine lyase.
  • sulfide compounds such as dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide are generated enzymatically or oxidatively from methyl mercaptan and hydrogen sulfide, which also cause bad odor.
  • Patent Document 1 discloses that a fragrance component is added as a deodorant or fragrance to a composition for cleaning, sterilizing, antifouling, and deodorizing a drain outlet or the like.
  • Patent Document 2 the action of an enzyme that generates methyl mercaptan from methionine or an enzyme that generates hydrogen sulfide from cysteine is inhibited by a specific fragrance component and is caused by a volatile sulfur compound generated from a waste or a drain outlet. It is disclosed to suppress malodors.
  • 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.
  • Patent Documents 3 to 4 disclose searching for substances that suppress malodor such as hexanoic acid and skatole, using as an index the activity of olfactory receptors that specifically respond to those malodorous substances.
  • Patent Document 5 discloses searching for a substance that suppresses sweat odor, using as an index the activity of an olfactory receptor that responds to a specific carboxylic acid.
  • Patent Document 6 discloses a method for identifying an agent 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.
  • Patent Document 7 discloses 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.
  • Patent Document 1 JP-A-2006-206882 (Patent Document 2) JP-A-2010-004971 (Patent Document 3) JP-A-2012-050411 (Patent Document 4) JP-A-2012-249614 (Patent Document 3) Literature 5) US Patent Publication No. 2013/0336910 (Patent Literature 6) International Publication No. 2006/094704 (Patent Literature 7) Japanese Translation of PCT International Publication No. 2004-504010
  • the present invention provides a method for evaluating and / or selecting a sulfide compound odor inhibitor comprising: Adding a test substance and a sulfide compound to at least one olfactory receptor polypeptide selected from the group consisting of OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with OR4S2; and Measuring the response of the olfactory receptor polypeptide to the sulfide compound;
  • a method comprising:
  • A Response to dimethyl disulfide
  • B Response to dimethyl trisulfide
  • DMDS Dimethyl disulfide
  • DMTS Dimethyl trisulfide
  • DMDS DMDS only
  • DMTS DMTS only
  • Vehicle mineral oil only
  • Error bar SE.
  • Wastes such as drain outlets and garbage generate various volatile sulfur compounds due to bacterial decay and cause bad odor.
  • One of the main causative substances of the malodor is sulfide compounds such as dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Therefore, control of the odor of these sulfide compounds is required.
  • the present inventor succeeded in newly identifying an olfactory receptor that specifically responds to a sulfide compound that is a causative substance of malodor from wastes such as drains and garbage.
  • the present inventor evaluates and / or evaluates substances that can suppress the odor of sulfide compounds by olfactory receptor antagonism by using the response of the olfactory receptor or a polypeptide having the same function as an index. It was found that selection is possible.
  • “inhibition of odor by olfactory receptor antagonism” means that by applying both the target odor molecule and another molecule, the other molecule inhibits the receptor response to the target odor molecule. As a result, it is a means for suppressing the odor recognized by the individual. Suppression of odor by olfactory receptor antagonism is distinguished from means for concealing the target odor by the fragrance of a fragrance, such as deodorization by a fragrance, even if the means uses other molecules.
  • An example of odor suppression by olfactory receptor antagonism is the case of using a substance that inhibits the response of olfactory receptors such as an antagonist (antagonist). If a substance that inhibits the response is applied to the receptor of the odor molecule that brings about a specific odor, the response of the receptor to the odor molecule is suppressed, so the odor finally perceived by the individual is suppressed. Can do.
  • 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 G ⁇ s, which can be expressed on the cell membrane and activated by binding of odor molecules (Nat. Neurosci., 2004, 5: 263-278).
  • 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), perform analysis with unit size to compare (ktup) as 2. Is calculated by
  • “at least 80% identity” with respect to nucleotide and amino acid sequences refers to 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably It means 98% or more, preferably 99% or more identity.
  • the “sulfide compound” refers to a compound represented by the following formula (I).
  • R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms.
  • Examples of the linear or branched alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, and Hexyl is mentioned.
  • Examples of the straight-chain or branched alkenyl group having 1 to 6 carbon atoms include vinyl, propenyl, allyl, butenyl, and methylbutenyl.
  • R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms, and the linear or branched chain having 1 to 4 carbon atoms
  • alkyl or alkenyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, and butenyl.
  • R 1 and R 2 are the same or different and are methyl, ethyl, propyl or isopropyl, and more preferably R 1 and R 2 are methyl.
  • n represents an integer of 1 to 5, preferably an integer of 1 to 3, and more preferably 2 or 3.
  • the “sulfide compound” is a volatile substance having a structure represented by the formula (I). More preferable examples of the “sulfide compound” include dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), allyl methyl sulfide, trimethyl sulfide, and the like, and more preferable examples include DMDS. And DMTS.
  • the “sulphide compound odor” suppressed by the present invention is an odor generated by the “sulfide compound” described above, and is preferably an odor generated by DMDS or DMTS.
  • the “sulphide compound odor” suppressed by the present invention may be a malodor emitted from spoiled garbage, sewage, or a drain.
  • OR4S2 As shown in FIGS. 1 and 2, the present inventor has identified OR4S2 as an olfactory receptor that specifically responds to a sulfide compound among many olfactory receptors. OR4S2 responds to various sulfide compounds in a concentration-dependent manner. Therefore, a substance that suppresses the response of OR4S2 or a polypeptide having a similar function causes a change in the recognition of the odor of the sulfide compound based on olfactory receptor antagonism, and as a result, the odor of the sulfide compound is changed. It can be selectively suppressed.
  • the present invention provides a method for evaluating and / or selecting a sulfide compound odor inhibitor.
  • the method comprises adding a test substance and a sulfide compound to at least one olfactory receptor polypeptide selected from the group consisting of OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with OR4S2; and Measuring the response of the olfactory receptor polypeptide to the sulfide compound.
  • a test substance that suppresses the response of the olfactory receptor polypeptide is identified based on the measured response.
  • the identified test substance is selected as an inhibitor of sulfide compound odor.
  • the method of the present invention may be a method performed in vitro or ex vivo.
  • a test substance and the sulfide compound are added to the olfactory receptor polypeptide having responsiveness to the sulfide compound.
  • 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 sulfide compound 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 OR4S2 and polypeptides having at least 80% identity in amino acid sequence with OR4S2.
  • OR4S2 is an olfactory receptor expressed in human olfactory cells.
  • OR4S2 is registered in GenBank as GI: 116517324.
  • OR4S2 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, encoded by the gene having the nucleotide sequence represented by SEQ ID NO: 1.
  • the polypeptide having at least 80% identity in the amino acid sequence with OR4S2 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, more preferably 98% or more, and still more preferably 99% or more, and responsiveness to the sulfide compound.
  • the olfactory receptor polypeptide used in the method of the present invention may be at least one selected from the olfactory receptor polypeptides described above, but may be a combination of any two or more.
  • OR4S2 is used.
  • the olfactory receptor polypeptide can be used in any form as long as it does not lose responsiveness to the sulfide compound.
  • 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.
  • the olfactory receptor polypeptide is a cell that naturally expresses the olfactory receptor polypeptide, such as a mammalian olfactory cell, or genetically engineered to express the olfactory receptor polypeptide. It can be a recombinant cell, or a culture thereof. Preferred examples include recombinant human cells that have been genetically engineered to express the olfactory receptor polypeptide. The recombinant cell can be produced by transforming the cell using a vector incorporating a gene encoding 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.
  • a gene encoding RTP1S is introduced into a cell together with a gene encoding the olfactory receptor polypeptide.
  • RTP1S includes human RTP1S.
  • Human RTP1S 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 registered in GenBank as GI: 50234917.
  • the response of the olfactory receptor polypeptide to the sulfide compound 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.
  • 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 G ⁇ s (Nat. Neurosci., 2004, 5: 263-278).
  • 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.
  • Yet another method includes measurement by electrophysiological techniques.
  • a cell such as Xenopus oocyte
  • the olfactory receptor polypeptide is co-expressed with other ion channels
  • the activity of the ion channel on the cell is measured by a patch clamp method
  • the response of the olfactory receptor polypeptide is measured by measuring with a two-electrode membrane potential fixation method or the like.
  • the test substance that suppresses the response can be 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 the sulfide compound to the response to the sulfide compound in the 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.
  • the olfactory receptor polypeptide before the treatment, cells not expressing the olfactory receptor polypeptide, and the like can be mentioned.
  • 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 test substance is a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound Can be identified as
  • the test substance When the response in the test substance addition group is suppressed as compared with the control group, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound.
  • the response of the olfactory receptor polypeptide in the test substance addition group measured by the above procedure is preferably 60% or less, more preferably 50% or less, and even more preferably 25% or less compared to the control group. If suppressed, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound.
  • the test substance is added to the sulfide compound. It can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to.
  • the test substance identified by the above procedure is a substance that can suppress the recognition of the odor of the sulfide compound by the individual by suppressing the response of the olfactory receptor to the sulfide compound. Therefore, the test substance identified by the above procedure can be selected as an inhibitor of sulfide compound odor.
  • the substance selected as the odor suppressor for sulfide compounds by the method of the present invention can suppress the odor of sulfide compounds by suppressing the response of olfactory receptors to sulfide compounds.
  • the substance selected by the method of the present invention may be an active ingredient of a sulfide compound odor inhibitor.
  • the substance selected by the method of this invention can be contained in the compound or composition for suppressing the smell of a sulfide compound as an active ingredient for suppressing the smell of a sulfide compound.
  • the substance selected by the method of the present invention can be used for the manufacture of a sulfide compound odor suppressor or for the manufacture of a compound or composition for suppressing the odor of a sulfide compound. .
  • the sulfide compound is represented by the following formula (I): R 1- [S] n -R 2 (I) (In the formula, R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5)
  • the method of [1] which is a compound represented by these.
  • R 1 and R 2 are Preferably, they are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms, More preferably, it is the same or different and represents methyl, ethyl, propyl or isopropyl, More preferably, R 1 and R 2 represent methyl, [2] The method according to the above.
  • n is preferably an integer of 1 to 3, more preferably 2 or 3.
  • OR4S2 is preferably a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
  • the polypeptide having at least 80% identity in amino acid sequence with OR4S2 is preferably 80% or more, more preferably 85% or more, and still more preferably, with the amino acid sequence represented by SEQ ID NO: 2. 90% or more, still more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more of an amino acid sequence, and a polypeptide having responsiveness to the sulfide compound.
  • the method according to any one of [1] to [7].
  • the method further comprises measuring a response to the sulfide compound in a 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; The olfactory receptor polypeptide before addition of the test substance; or a cell in which the olfactory receptor polypeptide is not expressed, Is that way.
  • test substance is added to the test substance. Identifying as a substance that suppresses the response of the olfactory receptor polypeptide to sulfide compounds; Further comprising a method.
  • the response of the olfactory receptor polypeptide to which the test substance is added to the sulfide compound is preferably 60% or less, more preferably 50% or less, and even more preferably 25% with respect to the response to the sulfide compound in the control group. Identifying the test substance as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound, if suppressed to: Further comprising a method.
  • At least one olfactory receptor polypeptide selected from the group consisting of the OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with the OR4S2 expresses the olfactory receptor polypeptide
  • 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, it is a cell into which a gene encoding the olfactory receptor polypeptide and a gene encoding human RTP1S are introduced.
  • At least one olfactory receptor polypeptide selected from the group consisting of the OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with the OR4S2, the above [14] or [15] The method according to any one of [1] to [13], wherein the recombinant cell or culture thereof is used.
  • the measurement of the response of the olfactory receptor polypeptide is measurement of intracellular cAMP amount by ELISA or reporter gene assay, measurement by calcium imaging, or electrophysiological measurement.
  • Example 1 Identification of Olfactory Receptor Responsive to Sulfide Compound (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.
  • pENTR vector Invitrogen
  • HEK293 cells expressing any one of 428 human olfactory receptors were prepared.
  • a reaction solution having the composition shown in Table 2 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).
  • HEK293 cells (3 ⁇ 10 5 cells / cm 2 ) were seeded in 90 ⁇ L each well, and cultured for 24 hours in an incubator maintained at 37 ° C. and 5% CO 2 .
  • cells (Mock) under conditions that do not express olfactory receptors were also prepared and used in the experiment.
  • Luciferase assay The olfactory receptor expressed in HEK293 cells is coupled with intracellular G ⁇ s to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP.
  • a luciferase reporter gene assay was used to monitor the increase in intracellular cAMP level as a luminescence value derived from the firefly luciferase gene (fluc2P-CRE-hygro).
  • a renilla luciferase gene fused with the CMV promoter downstream was simultaneously introduced, 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 ⁇ L of a solution containing a sulfide compound prepared with DMEM (Nacalai) was added.
  • the sulfide compound was dimethyl disulfide (DMDS) or dimethyl trisulfide (DMTS) 100 ⁇ M.
  • the cells were cultured for 3 hours in a CO 2 incubator to fully express the luciferase gene in the cells.
  • the luciferase activity was measured using a Dual-Glo TM luciferase assay system (Promega) according to the operation manual of the product.
  • a value fLuc / hRluc obtained by dividing the luminescence value derived from firefly luciferase by the luminescence value derived from Renilla luciferase was calculated.
  • a value obtained by dividing fLuc / hRluc derived from firefly luciferase induced by sulfide compound stimulation by fLuc / hRluc in cells not subjected to sulfide compound stimulation was calculated as a fold increase and used as an index of response intensity.
  • OR4S2 exhibits a specific response to both DMDS and DMTS (FIG. 1). ).
  • the response of OR4S2 to DMDS and DMTS was concentration dependent (FIG. 2).
  • OR4S2 also responded to dimethyl sulfide (DMS) at concentrations of 1 mM and 3 mM (FIG. 3).
  • DMS dimethyl sulfide
  • OR4S2 did not respond to 3 mM methyl mercaptan, which is also a volatile sulfur compound (FIG. 4). Therefore, OR4S2 is a sulfide compound receptor having responsiveness to various sulfide compounds.
  • OR4S2 is a novel sulfide compound receptor that has not been found to respond to sulfide compounds.
  • Example 2 Search for Sulfur Compound Odor Suppressor Based on Olfactory Receptor Response (1) Luciferase Assay HEK293 expressing OR4S2 (SEQ ID NO: 2) in the same procedure as in Examples 1 (1) to (3) Cells were made. According to the procedure of Example 1 (4), the response (fLuc / hRluc value) of the olfactory receptor to DMDS in the presence and absence of the test substance was measured by luciferase reporter gene assay.
  • fLuc / hRluc value (X ′) for DMTS single stimulation divided by fLuc / hRluc value (Y) of cells not subjected to DMTS stimulation (X ′ / Y) is DMTS response value (Fold). (increase).
  • the fLuc / hRluc value (Z ′) induced by co-stimulation of DMTS and the test substance was similarly compared with the fLuc / hRluc value (Y ) To calculate (Z ′ / Y) and set it as “Fold increment”.
  • the addition concentration of DMTS to the culture was 300 ⁇ M, and the addition concentration of the test substance was 100 ⁇ M.
  • Example 3 Inhibitory ability of sulfide compound odor by OR4S2 antagonist The inhibitory effect of sulfide compound odor by OR4S2 antagonist cis-4-heptenal identified in Example 2 and 1,4-cineole was confirmed by a sensory test. .
  • DMDS cis-4-heptenal, or 1,4-cineole
  • a 0.1% (v / v) solution of mineral oil was prepared.
  • DMTS a 0.01% (v / v) solution of mineral oil was prepared.
  • the glass bottle containing the cotton ball was covered and allowed to stand at 37 ° C. for 1 hour, and then used as a test sample for a sensory test.
  • a glass bottle containing only a cotton ball containing a solution of DMDS or DMTS as a reference sample and a glass bottle containing only a cotton ball containing mineral oil (Vehicle) as a target sample were prepared.
  • the sensory test was performed in a single blind manner by 10 evaluators for DMDS and 11 evaluators for DMTS. The test was started from 14:00 after 1 and a half hours had passed since the meal. In order to prevent the spread of odors, the test was basically performed near the draft. In order to eliminate the effect of adaptation to the odor of sulfide compounds, the perceived intensity of DMDS or DMTS odor was appropriately confirmed during the test, and a break was taken if necessary.
  • the evaluators were divided into two groups, one group in the order cis-4-heptenal and then 1,4-cineole, the other group in the order 1,4-cineole and then cis-4-heptenal in DMDS or The inhibitory effect of DMTS odor was evaluated. The test sample was replaced with a new one after evaluation by three evaluators.
  • each evaluator has the following five levels of criteria: “DMDS (or DMTS) odor is 1: unknown, 2: perceptible, 3: easy to understand, 4: feel strong, 5: endurable “I feel so strong” that the DMDS (or DMTS) odor intensity of DMDS (or DMTS) alone is 3, and the DMDS (or DMTS) odor intensity of each test sample in increments of 1.0 to 0.5.
  • the evaluation was made in nine stages up to zero. The average value of the evaluation results by each evaluator was obtained.
  • the results of the sensory test are shown in FIG.
  • the OR4S2 antagonists cis-4-heptenal and 1,4-cineole both suppressed the odor intensity of DMDS and DMTS. From the above results, it is clear that the odor of sulfide compounds such as DMDS and DMTS is suppressed by the OR4S2 antagonist, and therefore the odor inhibitor of sulfide compounds can be efficiently searched based on the response of OR4S2. It was done.

Abstract

The present invention identifies substances that suppress odors of sulfide compounds. Provided is a method for evaluating and/or selecting an agent for suppressing odors of sulfide compounds, the method comprising the following steps: adding a test substance and a sulfide compound to OR4S2 and at least one type of olfactory receptor polypeptide selected from among a group consisting of polypeptides having at least 80% identicality to OR4S2 in an amino acid sequence; and measuring the response of the olfactory receptor polypeptide to the sulfide compound.

Description

スルフィド化合物の臭いの抑制剤の評価又は選択方法Method for evaluating or selecting an inhibitor of sulfide compound odor
　本発明は、スルフィド化合物の臭いの抑制剤の評価又は選択方法に関する。 The present invention relates to a method for evaluating or selecting an inhibitor for odor of sulfide compounds.
　排水口や生ごみなどの廃棄物から発生する揮発性硫黄化合物は、低濃度でも人々に不快感を与える悪臭成分である。これらの揮発性硫黄化合物は、汚水や廃棄物などに含まれるシステインやメチオニンといった含硫黄アミノ酸もしくはそれらを含むタンパク質が、細菌がもつメチオニンリアーゼやシステインリアーゼなどの代謝酵素の作用で分解を受けることにより発生する。例えば、不快臭の原因となる揮発性硫黄化合物のうち、メチルメルカプタンは、メチオニンからメチオニンリアーゼの作用によって生成され、硫化水素は、システインからシステインリアーゼの作用によって生成される。さらにメチルメルカプタンや硫化水素からは、ジメチルスルフィド、ジメチルジスルフィド、ジメチルトリスルフィドなどのスルフィド化合物が酵素的あるいは酸化的に生成され、これらも悪臭の原因となる。 Volatile sulfur compounds generated from wastes such as drain outlets and garbage are malodorous components that make people uncomfortable even at low concentrations. These volatile sulfur compounds are produced by degradation of sulfur-containing amino acids such as cysteine and methionine contained in sewage and wastes or proteins containing them by the action of metabolic enzymes such as methionine lyase and cysteine lyase in bacteria. appear. For example, among volatile sulfur compounds that cause unpleasant odor, methyl mercaptan is produced from methionine by the action of methionine lyase, and hydrogen sulfide is produced from cysteine by the action of cysteine lyase. Further, sulfide compounds such as dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide are generated enzymatically or oxidatively from methyl mercaptan and hydrogen sulfide, which also cause bad odor.
　特許文献１には、排水口等の洗浄、殺菌、防汚、消臭用の組成物に、消臭剤又は芳香剤として香料成分を添加することが開示されている。特許文献２には、特定の香料成分により、メチオニンからメチルメルカプタンを生成する酵素、又はシステインから硫化水素を生成する酵素の作用を阻害し、廃棄物や排水口等から発生する揮発性硫黄化合物による悪臭を抑制することが開示されている。 Patent Document 1 discloses that a fragrance component is added as a deodorant or fragrance to a composition for cleaning, sterilizing, antifouling, and deodorizing a drain outlet or the like. According to Patent Document 2, the action of an enzyme that generates methyl mercaptan from methionine or an enzyme that generates hydrogen sulfide from cysteine is inhibited by a specific fragrance component and is caused by a volatile sulfur compound generated from a waste or a drain outlet. It is disclosed to suppress malodors.
　ヒト等の哺乳動物においては、匂いは、鼻腔上部の嗅上皮に存在する嗅神経細胞上の嗅覚受容体に匂い分子が結合し、それに対する受容体の応答が中枢神経系へと伝達されることにより認識されている。ヒトの場合、嗅覚受容体は約４００個存在することが報告されており、これらをコードする遺伝子はヒトの全遺伝子の約２％にあたる。一般的に、嗅覚受容体と匂い分子は複数対複数の組み合わせで対応付けられている。すなわち、個々の嗅覚受容体は構造の類似した複数の匂い分子を異なる親和性で受容し、一方で、個々の匂い分子は複数の嗅覚受容体によって受容される。さらに、ある嗅覚受容体を活性化する匂い分子が、別の嗅覚受容体の活性化を阻害するアンタゴニストとして働くことも報告されている。これら複数の嗅覚受容体の応答の組み合わせが、個々の匂いの認識をもたらしている。 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.
　したがって、同じ匂い分子が存在する場合でも、同時に他の匂い分子が存在すると、当該他の匂い分子によって受容体応答が阻害され、最終的に認識される匂いが全く異なることがある。このような仕組みを嗅覚受容体のアンタゴニズムと呼ぶ。この受容体アンタゴニズムによる匂いの抑制は、香水や芳香剤等の別の匂いを付加することによる消臭と異なり、特定の悪臭の認識を特異的に失くしてしまうことができ、また芳香剤の匂いによる不快感が生じることもないという利点を有している。 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. This antagonism suppression by receptor antagonism, unlike deodorization by adding another odor such as perfume or fragrance, can specifically lose recognition of specific malodors, and fragrance There is an advantage that there is no discomfort due to the smell.
　嗅覚受容体アンタゴニズムの考え方に基づき、嗅覚受容体の活性を指標として悪臭抑制物質を同定する方法がこれまでにいくつか開示されている。例えば、特許文献３～４には、ヘキサン酸やスカトール等の悪臭を抑制する物質を、それらの悪臭物質に特異的に応答する嗅覚受容体の活性を指標として探索することが開示されている。特許文献５には、特定のカルボン酸に応答する嗅覚受容体の活性を指標として、汗臭を抑制する物質を探索することが開示されている。特許文献６には、イソ吉草酸やその等価体の存在下で嗅覚受容体をコードするポリペプチドの活性を測定することで、当該ポリペプチドの機能を調節する薬剤を同定する方法が開示されている。特許文献７には、嗅覚受容体をコードするポリペプチドに特異的に結合する化合物を同定することにより、嗅覚の感覚に関連する化合物について化学物質のライブラリーをスクリーニングする方法が開示されている。 Based on the concept of olfactory receptor antagonism, several methods have been disclosed so far for identifying malodor-inhibiting substances using olfactory receptor activity as an index. For example, Patent Documents 3 to 4 disclose searching for substances that suppress malodor such as hexanoic acid and skatole, using as an index the activity of olfactory receptors that specifically respond to those malodorous substances. Patent Document 5 discloses searching for a substance that suppresses sweat odor, using as an index the activity of an olfactory receptor that responds to a specific carboxylic acid. Patent Document 6 discloses a method for identifying an agent 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 7 discloses 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.
　しかしながら、嗅覚受容体アンタゴニズムに基づいて、上記廃棄物や排水口等から発生する揮発性硫黄化合物の悪臭を抑える技術については、これまで報告がない。 However, there has been no report on the technology for suppressing the odor of volatile sulfur compounds generated from the wastes and drainage outlets based on the olfactory receptor antagonism.
　（特許文献１）特開２００６－２０６８８２号公報
　（特許文献２）特開２０１０－００４９７１号公報
　（特許文献３）特開２０１２－０５０４１１号公報
　（特許文献４）特開２０１２－２４９６１４号公報
　（特許文献５）米国特許公開第２０１３／０３３６９１０号
　（特許文献６）国際公開公報第２００６／０９４７０４号
　（特許文献７）特表２００４－５０４０１０号公報 (Patent Document 1) JP-A-2006-206882 (Patent Document 2) JP-A-2010-004971 (Patent Document 3) JP-A-2012-050411 (Patent Document 4) JP-A-2012-249614 (Patent Document 3) Literature 5) US Patent Publication No. 2013/0336910 (Patent Literature 6) International Publication No. 2006/094704 (Patent Literature 7) Japanese Translation of PCT International Publication No. 2004-504010
　一態様において、本発明は、スルフィド化合物の臭いの抑制剤の評価及び／又は選択方法であって、以下：
　ＯＲ４Ｓ２及びこれとアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドからなる群より選択される少なくとも１種の嗅覚受容体ポリペプチドに、試験物質及びスルフィド化合物を添加すること；及び、
　該スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を測定すること；
を含む方法を提供する。 In one aspect, the present invention provides a method for evaluating and / or selecting a sulfide compound odor inhibitor comprising:
Adding a test substance and a sulfide compound to at least one olfactory receptor polypeptide selected from the group consisting of OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with OR4S2; and
Measuring the response of the olfactory receptor polypeptide to the sulfide compound;
A method comprising:
嗅覚受容体のスルフィド化合物に対する応答。横軸は個々の嗅覚受容体、縦軸は応答強度を示す。データは３回の独立した実験からの平均値を示す。Ａ：ジメチルジスルフィドに対する応答、Ｂ：ジメチルトリスルフィドに対する応答。Response of olfactory receptors to sulfide compounds. The horizontal axis represents individual olfactory receptors, and the vertical axis represents response intensity. Data represent mean values from 3 independent experiments. A: Response to dimethyl disulfide, B: Response to dimethyl trisulfide. 種々の濃度のスルフィド化合物に対する嗅覚受容体ＯＲ４Ｓ２の応答。Ａ：ジメチルジスルフィド（ＤＭＤＳ）に対する応答、Ｂ：ジメチルトリスルフィド（ＤＭＴＳ）に対する応答。それぞれｎ＝３、エラーバー＝±ＳＥ。Response of the olfactory receptor OR4S2 to various concentrations of sulfide compounds. A: Response to dimethyl disulfide (DMDS), B: Response to dimethyl trisulfide (DMTS). N = 3 and error bar = ± SE, respectively. ジメチルスルフィドに対するＯＲ４Ｓ２の応答。それぞれｎ＝３、エラーバー＝±ＳＤ。Response of OR4S2 to dimethyl sulfide. N = 3 and error bar = ± SD, respectively. メチルメルカプタンに対するＯＲ４Ｓ２の応答。＋；試験物質あり（３ｍＭ）、－；試験物質なし。ｎ＝３、エラーバー＝±ＳＤ。Response of OR4S2 to methyl mercaptan. +: Test substance present (3 mM),-; No test substance. n = 3, error bar = ± SD. 各種試験物質による嗅覚受容体ＯＲ４Ｓ２のジメチルジスルフィド（ＤＭＤＳ）応答抑制効果。横軸は試験物質の濃度、縦軸はＤＭＤＳに対するＯＲ４Ｓ２の応答（Ｒｅｓｐｏｎｓｅ％）を示す。ｎ＝３、エラーバー＝±ＳＥ。Inhibitory effect of olfactory receptor OR4S2 on dimethyl disulfide (DMDS) response by various test substances. The horizontal axis represents the concentration of the test substance, and the vertical axis represents the OR4S2 response (Response%) to DMDS. n = 3, error bar = ± SE. 各種試験物質による嗅覚受容体ＯＲ４Ｓ２のジメチルトリスルフィド（ＤＭＴＳ）応答抑制効果。縦軸は試験物質存在下でのＤＭＴＳに対するＯＲ４Ｓ２の応答（ｆｏｌｄ　Ｉｎｃｒｅａｓｅ）を示す。－；試験物質なし。ｎ＝３、エラーバー＝±ＳＥ。Effect of olfactory receptor OR4S2 on dimethyltrisulfide (DMTS) response by various test substances. The vertical axis represents the OR4S2 response (fold Increase) to DMTS in the presence of the test substance. -; No test substance. n = 3, error bar = ± SE. 試験物質による悪臭抑制効果の官能評価結果。Ａ：ジメチルジスルフィド（ＤＭＤＳ）臭抑制効果、Ｂ：ジメチルトリスルフィド（ＤＭＴＳ）臭抑制効果。ＤＭＤＳ＝ＤＭＤＳのみ、ＤＭＴＳ＝ＤＭＴＳのみ、Ｖｅｈｉｃｌｅ＝ミネラルオイルのみ。ＤＭＤＳに関してｎ＝１０、ＤＭＴＳに関してｎ＝１１。エラーバー＝ＳＥ。Sensory evaluation results of odor control effect by test substance. A: Dimethyl disulfide (DMDS) odor suppressing effect, B: Dimethyl trisulfide (DMTS) odor suppressing effect. DMDS = DMDS only, DMTS = DMTS only, Vehicle = mineral oil only. N = 10 for DMDS and n = 11 for DMTS. Error bar = SE.
　排水口や生ごみなどの廃棄物からは、細菌による腐敗に伴い様々な揮発性硫黄化合物が発生し、悪臭の原因となる。その悪臭の主な原因物質の１種が、ジメチルスルフィド、ジメチルジスルフィド、ジメチルトリスルフィドなどのスルフィド化合物である。したがって、これらのスルフィド化合物の臭いの制御が求められている。 Wastes such as drain outlets and garbage generate various volatile sulfur compounds due to bacterial decay and cause bad odor. One of the main causative substances of the malodor is sulfide compounds such as dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Therefore, control of the odor of these sulfide compounds is required.
　本発明者は、排水口や生ごみなどの廃棄物からの悪臭の原因物質であるスルフィド化合物に特異的に応答する嗅覚受容体を新たに同定することに成功した。また本発明者は、当該嗅覚受容体又はそれと同様の機能を有するポリペプチドの応答を指標とすることにより、嗅覚受容体アンタゴニズムによってスルフィド化合物の臭いを抑制することができる物質の評価及び／又は選択が可能であることを見出した。 The present inventor succeeded in newly identifying an olfactory receptor that specifically responds to a sulfide compound that is a causative substance of malodor from wastes such as drains and garbage. In addition, the present inventor evaluates and / or evaluates substances that can suppress the odor of sulfide compounds by olfactory receptor antagonism by using the response of the olfactory receptor or a polypeptide having the same function as an index. It was found that selection is possible.
　本発明によれば、嗅覚受容体アンタゴニズムによりスルフィド化合物の臭いを選択的に消臭することができる物質を、効率よく探索することができる。 According to the present invention, it is possible to efficiently search for a substance that can selectively deodorize the odor of a sulfide compound by olfactory receptor antagonism.
　本明細書において、「嗅覚受容体アンタゴニズムによる臭いの抑制」とは、目的の臭い分子と他の分子をともに適用することにより、当該他の分子によって目的の臭い分子に対する受容体応答を阻害し、結果的に個体に認識される臭いを抑制する手段である。嗅覚受容体アンタゴニズムによる臭いの抑制は、同様に他の分子を用いる手段であっても、芳香剤による消臭のように、目的の臭いを香料の香気によって隠蔽する手段とは区別される。嗅覚受容体アンタゴニズムによる臭いの抑制の一例は、アンタゴニスト（拮抗剤）等の嗅覚受容体の応答を阻害する物質を使用するケースである。特定の臭いをもたらす臭い分子の受容体にその応答を阻害する物質を適用すれば、当該受容体の当該臭い分子に対する応答が抑制されるため、最終的に個体に知覚される臭いを抑制することができる。 In the present specification, “inhibition of odor by olfactory receptor antagonism” means that by applying both the target odor molecule and another molecule, the other molecule inhibits the receptor response to the target odor molecule. As a result, it is a means for suppressing the odor recognized by the individual. Suppression of odor by olfactory receptor antagonism is distinguished from means for concealing the target odor by the fragrance of a fragrance, such as deodorization by a fragrance, even if the means uses other molecules. An example of odor suppression by olfactory receptor antagonism is the case of using a substance that inhibits the response of olfactory receptors such as an antagonist (antagonist). If a substance that inhibits the response is applied to the receptor of the odor molecule that brings about a specific odor, the response of the receptor to the odor molecule is suppressed, so the odor finally perceived by the individual is suppressed. Can do.
　本明細書において、「嗅覚受容体ポリペプチド」とは、嗅覚受容体又はそれと同等の機能を有するポリペプチドをいい、嗅覚受容体と同等の機能を有するポリペプチドとは、嗅覚受容体と同様に、細胞膜上に発現することができ、匂い分子の結合によって活性化し、かつ活性化されると、細胞内のＧαｓと共役してアデニル酸シクラーゼを活性化することで細胞内ｃＡＭＰ量を増加させる機能を有するポリペプチドをいう（Ｎａｔ．Ｎｅｕｒｏｓｃｉ．，２００４，５：２６３－２７８）。 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).
　本明細書において、ヌクレオチド配列及びアミノ酸配列の同一性は、リップマン－パーソン法（Ｌｉｐｍａｎ－Ｐｅａｒｓｏｎ法；Ｓｃｉｅｎｃｅ，１９８５，２２７：１４３５－４１）によって計算される。具体的には、遺伝情報処理ソフトウェアＧｅｎｅｔｙｘ－Ｗｉｎ（Ｖｅｒ．５．１．１；ソフトウェア開発）のホモロジー解析（Ｓｅａｒｃｈ　ｈｏｍｏｌｏｇｙ）プログラムを用いて、Ｕｎｉｔ　ｓｉｚｅ　ｔｏ　ｃｏｍｐａｒｅ（ｋｔｕｐ）を２として解析を行うことにより算出される。 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), perform analysis with unit size to compare (ktup) as 2. Is calculated by
　本明細書において、ヌクレオチド配列及びアミノ酸配列に関する「少なくとも８０％の同一性」とは、８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上、さらにより好ましくは９８％以上、なお好ましくは９９％以上の同一性をいう。 As used herein, “at least 80% identity” with respect to nucleotide and amino acid sequences refers to 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably It means 98% or more, preferably 99% or more identity.
　本明細書において、「スルフィド化合物」とは、下記式（I）で表される化合物をいう。
　　R1－[S]n－R2　　　　(I) In the present specification, the “sulfide compound” refers to a compound represented by the following formula (I).
R 1- [S] n -R 2 (I)
　上記式（I）中、Ｒ1とＲ2は、同一又は異なって、炭素数１～６の直鎖又は分枝鎖のアルキル又はアルケニル基を示す。該炭素数１～６の直鎖又は分枝鎖のアルキル基の例としては、メチル、エチル、プロピル、イソプロピル、ブチル、イソブチル、sec-ブチル、tert-ブチル、ペンチル、イソペンチル、tert-ペンチル、及びヘキシルが挙げられる。該炭素数１～６の直鎖又は分枝鎖のアルケニル基の例としては、ビニル、プロペニル、アリル、ブテニル、及びメチルブテニルが挙げられる。好ましくは、該Ｒ1とＲ2は、同一又は異なって、炭素数１～４の直鎖又は分枝鎖のアルキル又はアルケニル基を示し、該炭素数１～４の直鎖又は分枝鎖のアルキル又はアルケニル基の例としては、メチル、エチル、プロピル、イソプロピル、ブチル、イソブチル、sec-ブチル、tert-ブチル、ビニル、プロペニル、アリル、及びブテニルが挙げられる。より好ましくは、該Ｒ1とＲ2は、同一又は異なって、メチル、エチル、プロピル又はイソプロピルであり、なお好ましくは、Ｒ1とＲ2はメチルである。 In the above formula (I), R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms. Examples of the linear or branched alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, and Hexyl is mentioned. Examples of the straight-chain or branched alkenyl group having 1 to 6 carbon atoms include vinyl, propenyl, allyl, butenyl, and methylbutenyl. Preferably, R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms, and the linear or branched chain having 1 to 4 carbon atoms Examples of alkyl or alkenyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, vinyl, propenyl, allyl, and butenyl. More preferably, R 1 and R 2 are the same or different and are methyl, ethyl, propyl or isopropyl, and more preferably R 1 and R 2 are methyl.
　上記式（I）中、ｎは、１～５の整数を示し、好ましくは１～３の整数を示し、より好ましくは２又は３を示す。 In the above formula (I), n represents an integer of 1 to 5, preferably an integer of 1 to 3, and more preferably 2 or 3.
　好ましくは、「スルフィド化合物」は、式（I）で表される構造を有する揮発性物質である。より好ましい「スルフィド化合物」の例としては、ジメチルスルフィド（ＤＭＳ）、ジメチルジスルフィド（ＤＭＤＳ）、ジメチルトリスルフィド（ＤＭＴＳ）、アリルメチルスルフィド、トリメチルスルフィドなどを挙げることができ、さらに好ましい例としては、ＤＭＤＳ及びＤＭＴＳが挙げられる。 Preferably, the “sulfide compound” is a volatile substance having a structure represented by the formula (I). More preferable examples of the “sulfide compound” include dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), allyl methyl sulfide, trimethyl sulfide, and the like, and more preferable examples include DMDS. And DMTS.
　本発明により抑制される「スルフィド化合物の臭い」とは、上述した「スルフィド化合物」により生じる臭いであり、好ましくは、ＤＭＤＳ又はＤＭＴＳにより生じる臭いである。代表的には、本発明により抑制される「スルフィド化合物の臭い」とは、腐敗した生ごみ、汚水、又は排水口から発せられる悪臭であり得る。 The “sulphide compound odor” suppressed by the present invention is an odor generated by the “sulfide compound” described above, and is preferably an odor generated by DMDS or DMTS. Typically, the “sulphide compound odor” suppressed by the present invention may be a malodor emitted from spoiled garbage, sewage, or a drain.
　図１～２に示すとおり、本発明者は、多くの嗅覚受容体の中から、スルフィド化合物に対して特異的に応答する嗅覚受容体としてＯＲ４Ｓ２を同定した。ＯＲ４Ｓ２は、各種スルフィド化合物に対して濃度依存的に応答する。したがって、ＯＲ４Ｓ２又はこれと同様の機能を有するポリペプチドの応答を抑制する物質は、嗅覚受容体アンタゴニズムに基づいて、スルフィド化合物の臭いの認識に変化を生じさせ、結果として、スルフィド化合物の臭いを選択的に抑制することができる。 As shown in FIGS. 1 and 2, the present inventor has identified OR4S2 as an olfactory receptor that specifically responds to a sulfide compound among many olfactory receptors. OR4S2 responds to various sulfide compounds in a concentration-dependent manner. Therefore, a substance that suppresses the response of OR4S2 or a polypeptide having a similar function causes a change in the recognition of the odor of the sulfide compound based on olfactory receptor antagonism, and as a result, the odor of the sulfide compound is changed. It can be selectively suppressed.
　したがって、本発明は、スルフィド化合物の臭いの抑制剤の評価及び／又は選択方法を提供する。当該方法は、ＯＲ４Ｓ２及びこれとアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドからなる群より選択される少なくとも１種の嗅覚受容体ポリペプチドに、試験物質及びスルフィド化合物を添加すること；及び、該スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を測定すること、を含む。測定された応答に基づいて該嗅覚受容体ポリペプチドの応答を抑制する試験物質が同定される。同定された試験物質は、スルフィド化合物の臭いの抑制剤として選択される。 Therefore, the present invention provides a method for evaluating and / or selecting a sulfide compound odor inhibitor. The method comprises adding a test substance and a sulfide compound to at least one olfactory receptor polypeptide selected from the group consisting of OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with OR4S2; and Measuring the response of the olfactory receptor polypeptide to the sulfide compound. A test substance that suppresses the response of the olfactory receptor polypeptide is identified based on the measured response. The identified test substance is selected as an inhibitor of sulfide compound odor.
　上記本発明の方法は、ｉｎ　ｖｉｔｒｏ又はｅｘ　ｖｉｖｏで行われる方法であり得る。本発明の方法においては、上記スルフィド化合物に応答性を有する嗅覚受容体ポリペプチドに、試験物質と該スルフィド化合物とが添加される。 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 the sulfide compound are added to the olfactory receptor polypeptide having responsiveness to the sulfide compound.
　本発明の方法に使用される試験物質は、スルフィド化合物の臭いの抑制剤として使用することを所望する物質であれば、特に制限されない。試験物質は、天然に存在する物質であっても、化学的若しくは生物学的方法等で人工的に合成した物質であってもよく、又は化合物であっても、組成物若しくは混合物であってもよい。 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 sulfide compound 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.
　本発明の方法に使用される嗅覚受容体ポリペプチドは、ＯＲ４Ｓ２、及びこれらとアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドからなる群より選択される少なくとも１種である。 The olfactory receptor polypeptide used in the method of the present invention is at least one selected from the group consisting of OR4S2 and polypeptides having at least 80% identity in amino acid sequence with OR4S2.
　ＯＲ４Ｓ２は、ヒト嗅細胞で発現している嗅覚受容体である。ＯＲ４Ｓ２は、ＧｅｎＢａｎｋにＧＩ：１１６５１７３２４として登録されている。ＯＲ４Ｓ２は、配列番号１で示されるヌクレオチド配列を有する遺伝子にコードされる、配列番号２で示されるアミノ酸配列からなるタンパク質である。 OR4S2 is an olfactory receptor expressed in human olfactory cells. OR4S2 is registered in GenBank as GI: 116517324. OR4S2 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, encoded by the gene having the nucleotide sequence represented by SEQ ID NO: 1.
　ＯＲ４Ｓ２とアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドとしては、配列番号２で示されるアミノ酸配列と少なくとも８０％、例えば８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上、さらにより好ましくは９８％以上、なお好ましくは９９％以上の同一性を有するアミノ酸配列からなり、かつ上記スルフィド化合物に応答性を有するポリペプチドが挙げられる。 The polypeptide having at least 80% identity in the amino acid sequence with OR4S2 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, more preferably 98% or more, and still more preferably 99% or more, and responsiveness to the sulfide compound.
　本発明の方法において使用される嗅覚受容体ポリペプチドは、上述した嗅覚受容体ポリペプチドから選択される少なくとも１種であればよいが、いずれか２種以上の組み合わせであってもよい。好ましくは、ＯＲ４Ｓ２が使用される。 The olfactory receptor polypeptide used in the method of the present invention may be at least one selected from the olfactory receptor polypeptides described above, but may be a combination of any two or more. Preferably OR4S2 is used.
　本発明の方法において、上記嗅覚受容体ポリペプチドは、上記スルフィド化合物に対する応答性を失わない限り、任意の形態で使用され得る。例えば、当該嗅覚受容体ポリペプチドは、生体から単離された嗅覚受容器若しくは嗅細胞等の、当該嗅覚受容体ポリペプチドを天然に発現する組織や細胞、又はそれらの培養物；当該嗅覚受容体ポリペプチドを担持した嗅細胞の膜；当該嗅覚受容体ポリペプチドを発現するように遺伝的に操作された組換え細胞又はその培養物；当該嗅覚受容体ポリペプチドを有する当該組換え細胞の膜；当該嗅覚受容体ポリペプチドを有する人工脂質二重膜、などの形態で使用され得る。これらの形態は全て、本発明で使用される嗅覚受容体ポリペプチドの範囲に含まれる。 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 the sulfide compound. 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.
　好ましい態様において、上記嗅覚受容体ポリペプチドは、哺乳動物の嗅細胞等の上記嗅覚受容体ポリペプチドを天然に発現する細胞、又は当該嗅覚受容体ポリペプチドを発現するように遺伝的に操作された組換え細胞、あるいはそれらの培養物であり得る。好ましい例としては、上記嗅覚受容体ポリペプチドを発現するように遺伝的に操作された組換えヒト細胞が挙げられる。当該組換え細胞は、当該嗅覚受容体ポリペプチドをコードする遺伝子を組み込んだベクターを用いて細胞を形質転換することで作製することができる。 In a preferred embodiment, the olfactory receptor polypeptide is a cell that naturally expresses the olfactory receptor polypeptide, such as a mammalian olfactory cell, or genetically engineered to express the olfactory receptor polypeptide. It can be a recombinant cell, or a culture thereof. Preferred examples include recombinant human cells that have been genetically engineered to express the olfactory receptor polypeptide. The recombinant cell can be produced by transforming the cell using a vector incorporating a gene encoding the olfactory receptor polypeptide.
　好適には、嗅覚受容体ポリペプチドの細胞膜発現を促進するために、当該嗅覚受容体ポリペプチドをコードする遺伝子とともに、ＲＴＰ（ｒｅｃｅｐｔｏｒ－ｔｒａｎｓｐｏｒｔｉｎｇ　ｐｒｏｔｅｉｎ）をコードする遺伝子を細胞に導入する。好ましくは、ＲＴＰ１Ｓをコードする遺伝子を、当該嗅覚受容体ポリペプチドをコードする遺伝子とともに細胞に導入する。ＲＴＰ１Ｓの例としては、ヒトＲＴＰ１Ｓが挙げられる。ヒトＲＴＰ１Ｓは、ＧｅｎＢａｎｋにＧＩ：５０２３４９１７として登録されている、配列番号３で示されるヌクレオチド配列を有する遺伝子にコードされた、配列番号４で示されるアミノ酸配列からなるタンパク質である。 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 an amino acid sequence represented by SEQ ID NO: 4 encoded by a gene having a nucleotide sequence represented by SEQ ID NO: 3 registered in GenBank as GI: 50234917.
　本発明の方法によれば、上記嗅覚受容体ポリペプチドへの試験物質及び上記スルフィド化合物の添加に続いて、該スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答が測定される。測定は、嗅覚受容体の応答を測定する方法として当該分野で知られている任意の方法、例えば、細胞内ｃＡＭＰ量測定等によって行えばよい。例えば、嗅覚受容体は、匂い分子によって活性化されると、細胞内のＧαｓと共役してアデニル酸シクラーゼを活性化することで、細胞内ｃＡＭＰ量を増加させることが知られている（Ｎａｔ．Ｎｅｕｒｏｓｃｉ．，２００４，５：２６３－２７８）。したがって、匂い分子添加後の細胞内ｃＡＭＰ量を指標にすることで、上記嗅覚受容体ポリペプチドの応答を測定することができる。ｃＡＭＰ量を測定する方法としては、ＥＬＩＳＡ法やレポータージーンアッセイ等が挙げられる。上記嗅覚受容体ポリペプチドの応答を測定する他の方法としては、カルシウムイメージング法が挙げられる。さらに別の方法としては、電気生理学的手法による測定が挙げられる。電気生理学的測定では、例えば、上記嗅覚受容体ポリペプチドを他のイオンチャネルとともに共発現させた細胞（アフリカツメガエル卵母細胞等）を作製し、該細胞上のイオンチャネルの活動をパッチクランプ法や二電極膜電位固定法などで測定することにより、該嗅覚受容体ポリペプチドの応答を測定する。 According to the method of the present invention, following the addition of the test substance and the sulfide compound to the olfactory receptor polypeptide, the response of the olfactory receptor polypeptide to the sulfide compound 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. Yet another method includes measurement by electrophysiological techniques. In electrophysiological measurement, for example, a cell (such as Xenopus oocyte) in which the olfactory receptor polypeptide is co-expressed with other ion channels is prepared, and the activity of the ion channel on the cell is measured by a patch clamp method, The response of the olfactory receptor polypeptide is measured by measuring with a two-electrode membrane potential fixation method or the like.
　さらに、測定された上記嗅覚受容体ポリペプチドの応答に基づいて、上記スルフィド化合物への応答に対して試験物質が及ぼす作用を評価すれば、当該応答を抑制する試験物質を同定することができる。試験物質による作用の評価は、試験物質を添加した該嗅覚受容体ポリペプチドの該スルフィド化合物に対する応答を、対照群における該スルフィド化合物に対する応答と比較することによって行うことができる。対照群としては、異なる濃度の試験物質を添加した該嗅覚受容体ポリペプチド、試験物質を添加しなかった該嗅覚受容体ポリペプチド、対照物質を添加した該嗅覚受容体ポリペプチド、試験物質を添加する前の該嗅覚受容体ポリペプチド、該嗅覚受容体ポリペプチドが発現していない細胞、などを挙げることができる。 Furthermore, if the action of the test substance on the response to the sulfide compound is evaluated based on the measured response of the olfactory receptor polypeptide, the test substance that suppresses the response can be 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 the sulfide compound to the response to the sulfide compound 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. The olfactory receptor polypeptide before the treatment, cells not expressing the olfactory receptor polypeptide, and the like can be mentioned.
　例えば、上記嗅覚受容体ポリペプチドの応答に対して試験物質が及ぼす作用は、より高濃度の試験物質添加群とより低濃度の試験物質添加群との間、試験物質添加群と非添加群との間、試験物質添加群と対照物質添加群との間、又は試験物質添加前後で、上記スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を比較することによって評価することができる。試験物質添加により、又はより高濃度の試験物質の添加により該嗅覚受容体ポリペプチドの応答が抑制される場合、当該試験物質を、該嗅覚受容体ポリペプチドの該スルフィド化合物に対する応答を抑制する物質として同定することができる。 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. , By comparing the response of the olfactory receptor polypeptide to the sulfide compound between the test substance addition group and the control substance addition group or before and after the test substance addition. When the response of the olfactory receptor polypeptide is suppressed by addition of a test substance or by addition of a higher concentration of the test substance, the test substance is a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound Can be identified as
　試験物質添加群における応答が、対照群よりも抑制されていた場合、試験物質を、上記嗅覚受容体ポリペプチドの上記スルフィド化合物に対する応答を抑制する物質として同定することができる。例えば、上記の手順で測定された試験物質添加群における上記嗅覚受容体ポリペプチドの応答が、対照群と比較して好ましくは６０％以下、より好ましくは５０％以下、さらに好ましくは２５％以下に抑制されていれば、当該試験物質を、上記スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を抑制する物質として同定することができる。あるいは、上記の手順で測定された試験物質添加群における上記嗅覚受容体ポリペプチドの応答が、対照群と比較して統計学的に有意に抑制されていれば、当該試験物質を、上記スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を抑制する物質として同定することができる。 When the response in the test substance addition group is suppressed as compared with the control group, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound. For example, the response of the olfactory receptor polypeptide in the test substance addition group measured by the above procedure is preferably 60% or less, more preferably 50% or less, and even more preferably 25% or less compared to the control group. If suppressed, the test substance can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound. 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 to the control group, the test substance is added to the sulfide compound. It can be identified as a substance that suppresses the response of the olfactory receptor polypeptide to.
　上記の手順で同定された試験物質は、スルフィド化合物に対する嗅覚受容体の応答を抑制することによって、個体によるスルフィド化合物の臭いの認識を抑制することができる物質である。したがって、上記手順で同定された試験物質は、スルフィド化合物の臭いの抑制剤として選択することができる。本発明の方法によってスルフィド化合物の臭いの抑制剤として選択された物質は、スルフィド化合物に対する嗅覚受容体の応答抑制によって、スルフィド化合物の臭いを抑制することができる。 The test substance identified by the above procedure is a substance that can suppress the recognition of the odor of the sulfide compound by the individual by suppressing the response of the olfactory receptor to the sulfide compound. Therefore, the test substance identified by the above procedure can be selected as an inhibitor of sulfide compound odor. The substance selected as the odor suppressor for sulfide compounds by the method of the present invention can suppress the odor of sulfide compounds by suppressing the response of olfactory receptors to sulfide compounds.
　したがって、一実施形態において、本発明の方法によって選択された物質は、スルフィド化合物の臭いの抑制剤の有効成分であり得る。あるいは、本発明の方法によって選択された物質は、スルフィド化合物の臭いを抑制するための化合物又は組成物に、スルフィド化合物の臭いを抑制するための有効成分として含有され得る。またあるいは、本発明の方法によって選択された物質は、スルフィド化合物の臭いの抑制剤の製造のため、又はスルフィド化合物の臭いを抑制するための化合物若しくは組成物の製造のために使用することができる。 Therefore, in one embodiment, the substance selected by the method of the present invention may be an active ingredient of a sulfide compound odor inhibitor. Or the substance selected by the method of this invention can be contained in the compound or composition for suppressing the smell of a sulfide compound as an active ingredient for suppressing the smell of a sulfide compound. Alternatively, the substance selected by the method of the present invention can be used for the manufacture of a sulfide compound odor suppressor or for the manufacture of a compound or composition for suppressing the odor of a sulfide compound. .
　本発明の例示的実施形態として、さらに以下の物質、製造方法、用途あるいは方法を本明細書に開示する。ただし、本発明はこれらの実施形態に限定されない。 As an exemplary embodiment of the present invention, the following substances, production methods, uses or methods are further disclosed herein. However, the present invention is not limited to these embodiments.
〔１〕スルフィド化合物の臭いの抑制剤の評価及び／又は選択方法であって、以下：
　ＯＲ４Ｓ２及びこれとアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドからなる群より選択される少なくとも１種の嗅覚受容体ポリペプチドに、試験物質及びスルフィド化合物を添加すること；及び、
　該スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を測定すること、
を含む方法。 [1] Evaluation and / or selection method of sulfide compound odor inhibitor,
Adding a test substance and a sulfide compound to at least one olfactory receptor polypeptide selected from the group consisting of OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with OR4S2; and
Measuring the response of the olfactory receptor polypeptide to the sulfide compound;
Including methods.
〔２〕好ましくは、上記スルフィド化合物が、下記式（I）：
　　R1－[S]n－R2　　　　(I)
（式中、Ｒ1とＲ2は、同一又は異なって、炭素数１～６の直鎖又は分枝鎖のアルキル又はアルケニル基を示し、ｎは１～５の整数を示す）
で表される化合物である、〔１〕記載の方法。 [2] Preferably, the sulfide compound is represented by the following formula (I):
R 1- [S] n -R 2 (I)
(In the formula, R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5)
The method of [1] which is a compound represented by these.
〔３〕上記Ｒ1とＲ2が、
　好ましくは、同一又は異なって、炭素数１～４の直鎖又は分枝鎖のアルキル又はアルケニル基を示し、
　より好ましくは、同一又は異なって、メチル、エチル、プロピル又はイソプロピルを示し、
　さらに好ましくは、Ｒ1とＲ2はメチルを示す、
〔２〕記載の方法。 [3] R 1 and R 2 are
Preferably, they are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms,
More preferably, it is the same or different and represents methyl, ethyl, propyl or isopropyl,
More preferably, R 1 and R 2 represent methyl,
[2] The method according to the above.
〔４〕上記ｎが、好ましくは１～３の整数を示し、より好ましくは２又は３を示す、〔２〕又は〔３〕記載の方法。 [4] The method according to [2] or [3], wherein n is preferably an integer of 1 to 3, more preferably 2 or 3.
〔５〕好ましくは、上記スルフィド化合物が、ジメチルスルフィド、ジメチルジスルフィド又はジメチルトリスルフィドである、〔１〕～〔４〕のいずれか１項記載の方法。 [5] The method according to any one of [1] to [4], wherein the sulfide compound is preferably dimethyl sulfide, dimethyl disulfide, or dimethyl trisulfide.
〔６〕好ましくは、上記スルフィド化合物の臭いが、腐敗した生ごみ、汚水、又は排水口から発せられる悪臭である、〔１〕～〔５〕のいずれか１項記載の方法。 [6] The method according to any one of [1] to [5], wherein the odor of the sulfide compound is preferably a malodor generated from spoiled garbage, sewage, or a drain.
〔７〕好ましくは、上記ＯＲ４Ｓ２が配列番号２で示されるアミノ酸配列からなるタンパク質である、〔１〕～〔６〕のいずれか１項記載の方法。 [7] The method according to any one of [1] to [6], wherein the OR4S2 is preferably a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
〔８〕好ましくは、上記ＯＲ４Ｓ２とアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドが、配列番号２で示されるアミノ酸配列と、好ましくは８０％以上、より好ましくは８５％以上、さらに好ましくは９０％以上、さらにより好ましくは９５％以上、なお好ましくは９８％以上、なおより好ましくは９９％以上の同一性を有するアミノ酸配列からなり、かつ上記スルフィド化合物に応答性を有するポリペプチドである、〔１〕～〔７〕のいずれか１項記載の方法。 [8] Preferably, the polypeptide having at least 80% identity in amino acid sequence with OR4S2 is preferably 80% or more, more preferably 85% or more, and still more preferably, with the amino acid sequence represented by SEQ ID NO: 2. 90% or more, still more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more of an amino acid sequence, and a polypeptide having responsiveness to the sulfide compound. The method according to any one of [1] to [7].
〔９〕好ましくは、測定された応答に基づいて該嗅覚受容体ポリペプチドの応答を抑制する試験物質を同定することをさらに含む、〔１〕～〔８〕のいずれか１項記載の方法。 [9] The method according to any one of [1] to [8], preferably further comprising identifying a test substance that suppresses the response of the olfactory receptor polypeptide based on the measured response.
〔１０〕上記〔１〕～〔８〕のいずれか１項記載の方法であって、
　好ましくは、対照群における上記スルフィド化合物に対する応答を測定することをさらに含み、かつ該対照群が、以下：
　上記試験物質を添加しなかった上記嗅覚受容体ポリペプチド；
　対照物質を添加した上記嗅覚受容体ポリペプチド；
　上記試験物質添加前の上記嗅覚受容体ポリペプチド；又は
　上記嗅覚受容体ポリペプチドが発現していない細胞、
である、方法。 [10] The method according to any one of [1] to [8] above,
Preferably, the method further comprises measuring a response to the sulfide compound in a 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;
The olfactory receptor polypeptide before addition of the test substance; or a cell in which the olfactory receptor polypeptide is not expressed,
Is that way.
〔１１〕上記〔１０〕記載の方法であって、好ましくは、
　上記試験物質を添加した上記嗅覚受容体ポリペプチドの上記スルフィド化合物に対する応答が、上記対照群における該スルフィド化合物に対する応答と比べて統計学的に有意に抑制されていた場合に、該試験物質を該スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を抑制する物質として同定すること、
をさらに含む、方法。 [11] The method according to [10] above, preferably,
When the response of the olfactory receptor polypeptide to which the test substance is added to the sulfide compound is statistically significantly suppressed as compared to the response to the sulfide compound in the control group, the test substance is added to the test substance. Identifying as a substance that suppresses the response of the olfactory receptor polypeptide to sulfide compounds;
Further comprising a method.
〔１２〕上記〔１０〕記載の方法であって、好ましくは、
　上記試験物質を添加した上記嗅覚受容体ポリペプチドの上記スルフィド化合物に対する応答が、上記対照群における該スルフィド化合物に対する応答に対して好ましくは６０％以下、より好ましくは５０％以下、さらに好ましくは２５％以下に抑制されていた場合に、該試験物質を該スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を抑制する物質として同定すること、
をさらに含む、方法。 [12] The method according to [10] above, preferably,
The response of the olfactory receptor polypeptide to which the test substance is added to the sulfide compound is preferably 60% or less, more preferably 50% or less, and even more preferably 25% with respect to the response to the sulfide compound in the control group. Identifying the test substance as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound, if suppressed to:
Further comprising a method.
〔１３〕好ましくは、上記スルフィド化合物に対する上記嗅覚受容体ポリペプチドの応答を抑制する試験物質を、スルフィド化合物の臭いの抑制剤として選択することをさらに含む、〔１〕～〔１２〕のいずれか１項記載の方法。 [13] Preferably, any one of [1] to [12], further comprising selecting a test substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound as an inhibitor of the odor of the sulfide compound. The method according to claim 1.
〔１４〕好ましくは、上記ＯＲ４Ｓ２及びこれとアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドからなる群より選択される少なくとも１種の嗅覚受容体ポリペプチドが、該嗅覚受容体ポリペプチドを発現するように遺伝的に操作された組換え細胞上に発現されている、〔１〕～〔１３〕のいずれか１項記載の方法。 [14] Preferably, at least one olfactory receptor polypeptide selected from the group consisting of the OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with the OR4S2 expresses the olfactory receptor polypeptide The method according to any one of [1] to [13], wherein the method is expressed on a genetically engineered recombinant cell.
〔１５〕上記〔１４〕記載の方法であって、上記組換え細胞が、
　好ましくは、上記嗅覚受容体ポリペプチドをコードする遺伝子と、ＲＴＰ１Ｓをコードする遺伝子とを導入された細胞であり、
　より好ましくは、上記嗅覚受容体ポリペプチドをコードする遺伝子と、ヒトＲＴＰ１Ｓをコードする遺伝子とを導入された細胞である、
方法。 [15] The method according to [14] 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, it is a cell into which a gene encoding the olfactory receptor polypeptide and a gene encoding human RTP1S are introduced.
Method.
〔１６〕好ましくは、上記ＯＲ４Ｓ２及びこれとアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドからなる群より選択される少なくとも１種の嗅覚受容体ポリペプチドとして、上記〔１４〕又は〔１５〕記載の組換え細胞又はその培養物が使用される、〔１〕～〔１３〕のいずれか１項記載の方法。 [16] Preferably, at least one olfactory receptor polypeptide selected from the group consisting of the OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with the OR4S2, the above [14] or [15] The method according to any one of [1] to [13], wherein the recombinant cell or culture thereof is used.
〔１７〕好ましくは、上記嗅覚受容体ポリペプチドの応答の測定が、ＥＬＩＳＡ若しくはレポータージーンアッセイによる細胞内ｃＡＭＰ量測定、カルシウムイメージングによる測定、又は電気生理学的測定である、〔１〕～〔１６〕のいずれか１項記載の方法。 [17] Preferably, the measurement of the response of the olfactory receptor polypeptide is measurement of intracellular cAMP amount by ELISA or reporter gene assay, measurement by calcium imaging, or electrophysiological measurement. The method of any one of Claims.
　以下、実施例を示し、本発明をより具体的に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
　本実施例で用いた臭い化合物及び試験物質を下記表１に示す。 The odorous compounds and test substances used in this example are shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
実施例１　スルフィド化合物に応答する嗅覚受容体の同定
（１）ヒト嗅覚受容体遺伝子のクローニング
　ヒト嗅覚受容体はＧｅｎＢａｎｋに登録されている配列情報を基に、ｈｕｍａｎ　ｇｅｎｏｍｉｃ　ＤＮＡ　ｆｅｍａｌｅ（Ｇ１５２１：Ｐｒｏｍｅｇａ）を鋳型としたＰＣＲ法によりクローニングした。ＰＣＲ法により増幅した各遺伝子をｐＥＮＴＲベクター（Ｉｎｖｉｔｒｏｇｅｎ）にマニュアルに従って組み込み、ｐＥＮＴＲベクター上に存在するＮｏｔＩ、ＡｓｃＩサイトを利用して、ｐＭＥ１８Ｓベクター上のＦｌａｇ－Ｒｈｏタグ配列の下流に作製したＮｏｔＩ、ＡｓｃＩサイトへと組換えた。 Example 1 Identification of Olfactory Receptor Responsive to Sulfide Compound (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）ｐＭＥ１８Ｓ－ヒトＲＴＰ１Ｓベクターの作製
　ＲＴＰ１Ｓ（配列番号４）をコードするＲＴＰ１Ｓ遺伝子（配列番号３）を、ｐＭＥ１８ＳベクターのＥｃｏＲＩ、ＸｈｏＩサイトへ組み込んだ。 (2) Preparation of pME18S-human RTP1S vector The RTP1S gene (SEQ ID NO: 3) encoding RTP1S (SEQ ID NO: 4) was incorporated into the EcoRI and XhoI sites of the pME18S vector.
（３）嗅覚受容体発現細胞の作製
　４２８種のヒト嗅覚受容体のいずれか１種を発現させたＨＥＫ２９３細胞を作製した。表２に示す組成の反応液を調製しクリーンベンチ内で１５分静置した後、９６ウェルプレート（ＢＤ）の各ウェルに添加した。次いで、ＨＥＫ２９３細胞（３×１０5細胞／ｃｍ2）を９０μＬずつ各ウェルに播種し、３７℃、５％ＣＯ2を保持したインキュベータ内で２４時間培養した。対照として用いるために、嗅覚受容体を発現させない条件の細胞（Ｍｏｃｋ）も用意し、同様に実験に用いた。 (3) Preparation of olfactory receptor-expressing cells HEK293 cells expressing any one of 428 human olfactory receptors were prepared. A reaction solution having the composition shown in Table 2 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). Subsequently, HEK293 cells (3 × 10 5 cells / cm 2 ) were seeded in 90 μL each well, and cultured for 24 hours in an incubator maintained at 37 ° C. and 5% CO 2 . For use as a control, cells (Mock) under conditions that do not express olfactory receptors were also prepared and used in the experiment.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（４）ルシフェラーゼアッセイ
　ＨＥＫ２９３細胞に発現させた嗅覚受容体は、細胞内在性のＧαｓと共役しアデニル酸シクラーゼを活性化することで、細胞内ｃＡＭＰ量を増加させる。本研究での匂い応答測定には、細胞内ｃＡＭＰ量の増加をホタルルシフェラーゼ遺伝子（ｆｌｕｃ２Ｐ－ＣＲＥ－ｈｙｇｒｏ）由来の発光値としてモニターするルシフェラーゼレポータージーンアッセイを用いた。また、ＣＭＶプロモータ下流にウミシイタケルシフェラーゼ遺伝子を融合させたもの（ｈＲｌｕｃ－ＣＭＶ）を同時に遺伝子導入し、遺伝子導入効率や細胞数の誤差を補正する内部標準として用いた。 (4) Luciferase assay The olfactory receptor expressed in HEK293 cells is coupled with intracellular Gαs to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP. For the measurement of odor response in this study, a luciferase reporter gene assay was used to monitor 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 with the CMV promoter downstream (hRluc-CMV) was simultaneously introduced, and used as an internal standard for correcting errors in gene transfer efficiency and cell number.
　上記（３）で作製した培養物から、培地を取り除き、ＤＭＥＭ（Ｎａｃａｌａｉ）で調製したスルフィド化合物を含む溶液を７５μＬ添加した。スルフィド化合物は、ジメチルジスルフィド（ＤＭＤＳ）又はジメチルトリスルフィド（ＤＭＴＳ）１００μＭとした。細胞をＣＯ2インキュベータ内で３時間培養し、ルシフェラーゼ遺伝子を細胞内で十分に発現させた。ルシフェラーゼの活性測定には、Ｄｕａｌ－ＧｌｏTMｌｕｃｉｆｅｒａｓｅ　ａｓｓａｙ　ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ）を用い、製品の操作マニュアルに従って測定を行った。各種刺激条件について、ホタルルシフェラーゼ由来の発光値をウミシイタケルシフェラーゼ由来の発光値で除した値ｆＬｕｃ／ｈＲｌｕｃを算出した。スルフィド化合物刺激により誘導されたホタルルシフェラーゼ由来のｆＬｕｃ／ｈＲｌｕｃを、スルフィド化合物刺激を行わない細胞でのｆＬｕｃ／ｈＲｌｕｃで割った値をｆｏｌｄ　ｉｎｃｒｅａｓｅとして算出し、応答強度の指標とした。 The medium was removed from the culture prepared in (3) above, and 75 μL of a solution containing a sulfide compound prepared with DMEM (Nacalai) was added. The sulfide compound was dimethyl disulfide (DMDS) or dimethyl trisulfide (DMTS) 100 μM. The cells were cultured for 3 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. For various stimulation conditions, a value fLuc / hRluc obtained by dividing the luminescence value derived from firefly luciferase by the luminescence value derived from Renilla luciferase was calculated. A value obtained by dividing fLuc / hRluc derived from firefly luciferase induced by sulfide compound stimulation by fLuc / hRluc in cells not subjected to sulfide compound stimulation was calculated as a fold increase and used as an index of response intensity.
（５）結果
　４２８種類の嗅覚受容体についてＤＭＤＳ又はＤＭＴＳに対する応答を測定した結果、嗅覚受容体ＯＲ４Ｓ２が、ＤＭＤＳ及びＤＭＴＳの両方に対して特異的な応答を示すことが明らかになった（図１）。ＯＲ４Ｓ２のＤＭＤＳ及びＤＭＴＳに対する応答は濃度依存的であった（図２）。またＯＲ４Ｓ２は、１ｍＭと３ｍＭの濃度のジメチルスルフィド（ＤＭＳ）に対しても応答した（図３）。一方で、ＯＲ４Ｓ２は、同じく揮発性硫黄化合物であるメチルメルカプタン３ｍＭに対しては応答しなかった（図４）。したがって、ＯＲ４Ｓ２は、様々なスルフィド化合物に対して応答性をもつスルフィド化合物受容体である。またＯＲ４Ｓ２は、これまでスルフィド化合物に応答することが見出されていない、新規のスルフィド化合物受容体である。 (5) Results As a result of measuring responses to DMDS or DMTS for 428 types of olfactory receptors, it was revealed that the olfactory receptor OR4S2 exhibits a specific response to both DMDS and DMTS (FIG. 1). ). The response of OR4S2 to DMDS and DMTS was concentration dependent (FIG. 2). OR4S2 also responded to dimethyl sulfide (DMS) at concentrations of 1 mM and 3 mM (FIG. 3). On the other hand, OR4S2 did not respond to 3 mM methyl mercaptan, which is also a volatile sulfur compound (FIG. 4). Therefore, OR4S2 is a sulfide compound receptor having responsiveness to various sulfide compounds. OR4S2 is a novel sulfide compound receptor that has not been found to respond to sulfide compounds.
実施例２　嗅覚受容体応答に基づくスルフィド化合物の臭いの抑制剤の探索
（１）ルシフェラーゼアッセイ
　実施例１（１）～（３）と同様の手順で、ＯＲ４Ｓ２（配列番号２）を発現させたＨＥＫ２９３細胞を作製した。実施例１（４）の手順に従って、ルシフェラーゼレポータージーンアッセイにより、試験物質存在下及び非存在下での嗅覚受容体のＤＭＤＳに対する応答（ｆＬｕｃ／ｈＲｌｕｃ値）を測定した。ＤＭＤＳ単独刺激により誘導されたｆＬｕｃ／ｈＲｌｕｃ値（Ｘ）、ＤＭＤＳ刺激を行わなかった細胞でのｆＬｕｃ／ｈＲｌｕｃ値（Ｙ）、ＤＭＤＳと試験物質との共刺激により誘導されたｆＬｕｃ／ｈＲｌｕｃ値（Ｚ）を求め、以下の計算式により、試験物質存在下での受容体のＤＭＤＳ応答強度（Ｒｅｓｐｏｎｓｅ（％））を求めた。
　　Ｒｅｓｐｏｎｓｅ（％）＝（Ｚ－Ｙ）／（Ｘ－Ｙ）×１００
独立した実験を３回行い、各回の実験の平均値を求めた。培養物へのＤＭＤＳの添加濃度は１ｍＭとし、試験物質の添加濃度は０～３０００μＭの範囲で変更した。 Example 2 Search for Sulfur Compound Odor Suppressor Based on Olfactory Receptor Response (1) Luciferase Assay HEK293 expressing OR4S2 (SEQ ID NO: 2) in the same procedure as in Examples 1 (1) to (3) Cells were made. According to the procedure of Example 1 (4), the response (fLuc / hRluc value) of the olfactory receptor to DMDS in the presence and absence of the test substance was measured by luciferase reporter gene assay. FLuc / hRluc value (X) induced by DMDS single stimulation, fLuc / hRluc value (Y) in cells not subjected to DMDS stimulation, fLuc / hRluc value induced by costimulation of DMDS and test substance (Z The DMDS response intensity (Response (%)) of the receptor in the presence of the test substance was determined by the following calculation formula.
Response (%) = (ZY) / (XY) × 100
Three independent experiments were performed, and the average value of each experiment was determined. The addition concentration of DMDS to the culture was 1 mM, and the addition concentration of the test substance was changed in the range of 0 to 3000 μM.
　ＤＭＴＳ応答については、ＤＭＴＳ単独刺激に対するｆＬｕｃ／ｈＲｌｕｃ値（Ｘ'）を、ＤＭＴＳ刺激を行わなかった細胞のｆＬｕｃ／ｈＲｌｕｃ値（Ｙ）で除した値（Ｘ'／Ｙ）をＤＭＴＳ応答値（Ｆｏｌｄ　ｉｎｃｒｅａｓｅ）として算出した。このＤＭＴＳ応答に対する試験物質の効果を比較するために、ＤＭＴＳと試験物質との共刺激により誘導されたｆＬｕｃ／ｈＲｌｕｃ値（Ｚ'）を同様に刺激を行わなかった細胞のｆＬｕｃ／ｈＲｌｕｃ値（Ｙ）で除して（Ｚ'／Ｙ）を算出し、Ｆｏｌｄ　ｉｎｃｒｅａｓｅとした。独立した実験を３回行い、各回の実験の平均値を求めた。培養物へのＤＭＴＳの添加濃度は３００μＭ、試験物質の添加濃度は１００μＭとした。 For DMTS response, fLuc / hRluc value (X ′) for DMTS single stimulation divided by fLuc / hRluc value (Y) of cells not subjected to DMTS stimulation (X ′ / Y) is DMTS response value (Fold). (increase). In order to compare the effect of the test substance on this DMTS response, the fLuc / hRluc value (Z ′) induced by co-stimulation of DMTS and the test substance was similarly compared with the fLuc / hRluc value (Y ) To calculate (Z ′ / Y) and set it as “Fold increment”. Three independent experiments were performed, and the average value of each experiment was determined. The addition concentration of DMTS to the culture was 300 μM, and the addition concentration of the test substance was 100 μM.
　結果を図５及び図６に示す。ｃｉｓ－４－ヘプテナール及び１，４－シネオールは、いずれも濃度依存的にＯＲ４Ｓ２のＤＭＤＳ応答を抑制した。またこれら２つの化合物はいずれも、ＯＲ４Ｓ２のＤＭＴＳ応答も抑制した。これらの結果から、上記２つの化合物が、ＯＲ４Ｓ２アンタゴニストであることが明らかにされた。 The results are shown in FIGS. Both cis-4-heptenal and 1,4-cineole suppressed the DMDS response of OR4S2 in a concentration-dependent manner. In addition, both of these two compounds also suppressed the DM4 response of OR4S2. From these results, it was revealed that the two compounds are OR4S2 antagonists.
実施例３　ＯＲ４Ｓ２アンタゴニストによるスルフィド化合物の臭いの抑制能
　実施例２で同定したＯＲ４Ｓ２アンタゴニストであるｃｉｓ－４－ヘプテナール、及び１，４－シネオールによるスルフィド化合物の臭いの抑制効果を、官能試験により確認した。 Example 3 Inhibitory ability of sulfide compound odor by OR4S2 antagonist The inhibitory effect of sulfide compound odor by OR4S2 antagonist cis-4-heptenal identified in Example 2 and 1,4-cineole was confirmed by a sensory test. .
　ＤＭＤＳ、ｃｉｓ－４－ヘプテナール、又は１，４－シネオールについては、ミネラルオイルの０．１％（ｖ／ｖ）溶液を調製した。ＤＭＴＳについては、ミネラルオイルの０．０１％（ｖ／ｖ）溶液を調製した。２０ｍＬ容のガラス瓶（マルエム、Ｎｏ．６）に２つの綿球を入れ、１つの綿球にはＤＭＤＳ又はＤＭＴＳの溶液３０μＬを、もう１つの綿球には上記ＯＲ４Ｓ２アンタゴニストのいずれかの溶液３０μＬを染み込ませた。上記綿球を入れたガラス瓶は、蓋をして３７℃で１時間静置した後、試験サンプルとして官能試験に用いた。基準サンプルとしてＤＭＤＳ又はＤＭＴＳの溶液を含む綿球のみを入れたガラス瓶を、対象サンプルとしてミネラルオイル（Ｖｅｈｉｃｌｅ）を含む綿球のみを入れたガラス瓶を準備した。 For DMDS, cis-4-heptenal, or 1,4-cineole, a 0.1% (v / v) solution of mineral oil was prepared. For DMTS, a 0.01% (v / v) solution of mineral oil was prepared. Place two cotton balls in a 20 mL glass bottle (Marem, No. 6), one cotton ball with 30 μL of DMDS or DMTS solution and the other cotton ball with 30 μL of any of the above OR4S2 antagonist solutions. Soaked. The glass bottle containing the cotton ball was covered and allowed to stand at 37 ° C. for 1 hour, and then used as a test sample for a sensory test. A glass bottle containing only a cotton ball containing a solution of DMDS or DMTS as a reference sample and a glass bottle containing only a cotton ball containing mineral oil (Vehicle) as a target sample were prepared.
　官能試験は、ＤＭＤＳに関しては１０名、ＤＭＴＳに関しては１１名の評価者により単盲式にて行った。試験は、食後１時間半以上を経過した１４時以降から開始した。匂いの拡散を防ぐため、試験は基本的にドラフト付近で行った。スルフィド化合物の臭いへの順応の影響を排除するため、試験中には適宜、ＤＭＤＳ又はＤＭＴＳ臭の認知強度を確認させ、必要であれば休憩をとった。評価者を二群に分け、一方の群はｃｉｓ－４－ヘプテナール、次いで１，４－シネオールの順序で、他方の群は１、４－シネオール、次いでｃｉｓ－４－ヘプテナールの順序で、ＤＭＤＳ又はＤＭＴＳ臭の抑制効果を評価した。試験サンプルは３名の評価者による評価後に新しいものと交換した。
　試験サンプルの臭い評価では、各評価者に、次の５段階の基準「ＤＭＤＳ（又はＤＭＴＳ）の臭いが、１：わからない、２：感知できる、３：楽にわかる、４：強く感じる、５：耐えられないほど強く感じる」を設定し、ＤＭＤＳ（又はＤＭＴＳ）単独での臭い強度を３としたときの各試験サンプルのＤＭＤＳ（又はＤＭＴＳ）臭の強度を１．０から０．５刻みで５．０までの９段階で評価させた。各評価者による評価結果の平均値を求めた。 The sensory test was performed in a single blind manner by 10 evaluators for DMDS and 11 evaluators for DMTS. The test was started from 14:00 after 1 and a half hours had passed since the meal. In order to prevent the spread of odors, the test was basically performed near the draft. In order to eliminate the effect of adaptation to the odor of sulfide compounds, the perceived intensity of DMDS or DMTS odor was appropriately confirmed during the test, and a break was taken if necessary. The evaluators were divided into two groups, one group in the order cis-4-heptenal and then 1,4-cineole, the other group in the order 1,4-cineole and then cis-4-heptenal in DMDS or The inhibitory effect of DMTS odor was evaluated. The test sample was replaced with a new one after evaluation by three evaluators.
In the odor evaluation of the test sample, each evaluator has the following five levels of criteria: “DMDS (or DMTS) odor is 1: unknown, 2: perceptible, 3: easy to understand, 4: feel strong, 5: endurable “I feel so strong” that the DMDS (or DMTS) odor intensity of DMDS (or DMTS) alone is 3, and the DMDS (or DMTS) odor intensity of each test sample in increments of 1.0 to 0.5. The evaluation was made in nine stages up to zero. The average value of the evaluation results by each evaluator was obtained.
　官能試験の結果を図７に示す。ＯＲ４Ｓ２アンタゴニストであるｃｉｓ－４－ヘプテナール及び１，４－シネオールは、いずれもＤＭＤＳ及びＤＭＴＳの臭い強度を抑制した。以上の結果から、ＯＲ４Ｓ２アンタゴニストにより、ＤＭＤＳやＤＭＴＳなどのスルフィド化合物の臭いが抑制されること、したがって、ＯＲ４Ｓ２の応答に基づいてスルフィド化合物の臭いの抑制剤を効率よく探索することができることが明らかにされた。 The results of the sensory test are shown in FIG. The OR4S2 antagonists cis-4-heptenal and 1,4-cineole both suppressed the odor intensity of DMDS and DMTS. From the above results, it is clear that the odor of sulfide compounds such as DMDS and DMTS is suppressed by the OR4S2 antagonist, and therefore the odor inhibitor of sulfide compounds can be efficiently searched based on the response of OR4S2. It was done.

Claims (8)

  1. 　スルフィド化合物の臭いの抑制剤の評価及び／又は選択方法であって、以下：
    　ＯＲ４Ｓ２及びこれとアミノ酸配列において少なくとも８０％の同一性を有するポリペプチドからなる群より選択される少なくとも１種の嗅覚受容体ポリペプチドに、試験物質及びスルフィド化合物を添加すること；及び、
    　該スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を測定すること、
    を含む方法。     A method for evaluating and / or selecting a sulfide compound odor inhibitor comprising:
    Adding a test substance and a sulfide compound to at least one olfactory receptor polypeptide selected from the group consisting of OR4S2 and a polypeptide having at least 80% identity in amino acid sequence with OR4S2; and
    Measuring the response of the olfactory receptor polypeptide to the sulfide compound;
    Including methods.
  2. 　前記スルフィド化合物が、下記式（I）：
    　　R1－[S]n－R2　　　　(I)
    （式中、Ｒ1とＲ2は、同一又は異なって、炭素数１～６の直鎖又は分枝鎖のアルキル又はアルケニル基を示し、ｎは１～５の整数を示す）
    で表される化合物である、請求項１記載の方法。     The sulfide compound is represented by the following formula (I):
    R 1- [S] n -R 2 (I)
    (In the formula, R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5)
    The method of Claim 1 which is a compound represented by these.
  3. 　前記Ｒ1とＲ2が、同一又は異なって、炭素数１～４の直鎖又は分枝鎖のアルキル又はアルケニル基であり、ｎは１～３の整数である、請求項２記載の方法。 The method according to claim 2, wherein R 1 and R 2 are the same or different and each represents a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms, and n is an integer of 1 to 3.
  4. 　前記ＯＲ４Ｓ２が配列番号２で示されるアミノ酸配列からなるタンパク質である、請求項１～３のいずれか１項記載の方法。 The method according to any one of claims 1 to 3, wherein the OR4S2 is a protein comprising an amino acid sequence represented by SEQ ID NO: 2.
  5. 　前記嗅覚受容体ポリペプチドが、該嗅覚受容体ポリペプチドを発現するように遺伝的に操作された組換え細胞上に発現されている、請求項１～４のいずれか１項記載の方法。 The method according to any one of claims 1 to 4, wherein the olfactory receptor polypeptide is expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide.
  6. 　前記試験物質を添加しなかった前記嗅覚受容体ポリペプチドのスルフィド化合物に対する応答を測定することをさらに含む、請求項１～５のいずれか１項記載の方法。 The method according to any one of claims 1 to 5, further comprising measuring a response of the olfactory receptor polypeptide to which the test substance is not added to a sulfide compound.
  7. 　前記試験物質を添加した前記嗅覚受容体ポリペプチドの前記スルフィド化合物に対する応答が、該試験物質を添加しなかった該嗅覚受容体ポリペプチドの該スルフィド化合物に対する応答よりも抑制されていたときに、該試験物質を、該スルフィド化合物に対する該嗅覚受容体ポリペプチドの応答を抑制する物質として同定することをさらに含む、請求項６記載の方法。 When the response of the olfactory receptor polypeptide to which the test substance is added to the sulfide compound is suppressed more than the response of the olfactory receptor polypeptide to which the test substance is not added to the sulfide compound, 7. The method of claim 6, further comprising identifying a test substance as a substance that suppresses the response of the olfactory receptor polypeptide to the sulfide compound.
  8. 　前記嗅覚受容体ポリペプチドの応答の測定が、ＥＬＩＳＡ若しくはレポータージーンアッセイによる細胞内ｃＡＭＰ量測定、カルシウムイメージングによる測定、又は電気生理学的測定である、請求項１～７のいずれか１項記載の方法。 The method according to any one of claims 1 to 7, wherein the measurement of the response of the olfactory receptor polypeptide is measurement of intracellular cAMP amount by ELISA or reporter gene assay, measurement by calcium imaging, or electrophysiological measurement.
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