WO2016194788A1 - Method for selecting odor-controlling substance - Google Patents

Method for selecting odor-controlling substance Download PDF

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Publication number
WO2016194788A1
WO2016194788A1 PCT/JP2016/065656 JP2016065656W WO2016194788A1 WO 2016194788 A1 WO2016194788 A1 WO 2016194788A1 JP 2016065656 W JP2016065656 W JP 2016065656W WO 2016194788 A1 WO2016194788 A1 WO 2016194788A1
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Prior art keywords
odor
olfactory receptor
substance
target
receptor polypeptide
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PCT/JP2016/065656
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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 JP2016096146A external-priority patent/JP6114439B2/en
Application filed by 花王株式会社 filed Critical 花王株式会社
Priority to EP16803223.3A priority Critical patent/EP3305898B1/en
Priority to US15/576,514 priority patent/US10738101B2/en
Publication of WO2016194788A1 publication Critical patent/WO2016194788A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • G01N2500/10Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a method for suppressing odor based on an odor adaptation mechanism.
  • odorous substances are recognized by olfactory receptors of olfactory neurons existing in the olfactory epithelium that spreads in the deepest part of the nasal cavity.
  • the odor molecules taken into the nasal cavity act on the olfactory receptor to activate it, and the signal from the olfactory nerve cell caused by the activated olfactory receptor is transmitted to the central nervous system. Is perceived.
  • sensory deodorization includes (1) the method of introducing other odorous substances into the malodorous environment and smelling them with the bad odor (masking), and (2) smelling other odorous substances before smelling the bad odor.
  • the method of sensory deodorization (2) is a method based on the physiological phenomenon of cross adaptation of olfaction.
  • the cross-adaptation of olfaction is defined as habituation to a different odor substance due to habituation to a certain odor substance and a decrease in olfactory sensitivity (Non-patent Document 3). That is, by getting used to odors that are not offensive odor, habituation occurs even for bad odors, and perception is suppressed.
  • Patent Document 2 an odor suppression method based on cross-adaptation that reduces the olfactory sensitivity to the odor-causing component by continuing to smell a substance that has a chemical structure that closely resembles the odor-causing component but has a weaker or unpleasant odor Is described.
  • Patent Document 3 describes a method for evaluating the odor similarity between a natural flavor and an imitation flavor simulating the odor based on cross adaptation. In Patent Document 3, assuming that two flavor compositions have the same odor, stronger cross adaptation occurs, and the effect of the adaptation appears in cerebral blood flow change. Evaluates the similarity of two flavors.
  • the decrease in odor sensitivity due to cross-adaptation can occur by various mechanisms such as olfactory receptor level, neural circuit level as well as olfactory receptor level.
  • Various hypotheses have been considered as the mechanism of olfactory cross-adaptation.
  • One is the hypothesis that cross-adaptation occurs in neurons in higher brain regions. In other words, it is a mechanism that loses sensitivity so that neurons in the higher brain region where information from peripheral olfactory neurons is integrated and odor quality is decoded do not continue to respond unnecessarily to odor information that remains unchanged .
  • Another is the hypothesis that cross-adaptation occurs at the level of olfactory receptors in peripheral olfactory neurons.
  • Non-patent Document 3 a substance causing cross-adaptation of a certain odor has a chemical structure very similar to that of the odor causing substance or a substance having a similar odor quality.
  • Non-Patent Document 4 it is reported that odor substances received by a single rat olfactory receptor have similar odor qualities, and these odor substances cause cross-adaptation in humans.
  • one odorant is recognized by many olfactory receptors with different selectivity.
  • Patent Literature 1 Japanese Patent No. 5646255 (Patent Literature 2) Japanese Patent Laid-Open No. 2005-53887 (Patent Literature 3) Japanese Patent No. 4966790 (Non-Patent Literature 1) Shirasu M. et al., Neuron, 81: 165 -178, 2014 (Non-Patent Document 2) venue JD et al., Nat.
  • Non-patent document 3 Kawasaki Michiaki and Horiuchi Tetsuro “Smell and smell substance” p71-72, Association of smell and smell, 1998 (Non-patent document 4) Pierce JD, Chemical senses, 21: 223-237 , 1996 (Non-Patent Document 5) Chemosensory Perception, 3 (3): 149-155, 2010
  • the present invention is a method for selecting a substance that causes cross-adaptation of a target odor, (1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent; (2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
  • a method comprising:
  • the present invention also provides a method for selecting a substance that suppresses the target odor, (1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent; (2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that suppresses the odor of the target;
  • a method comprising:
  • the present invention provides a method for selecting a substance that causes cross-adaptation of a target odor, (1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent; (2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
  • a method comprising:
  • the present invention is a method for selecting a substance that suppresses the odor of a target, (1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent; (2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that suppresses the target odor;
  • a method comprising:
  • Olfactory receptor response to muscone, guaiacol, p-cresol Olfactory receptor response to muscone, guaiacol, p-cresol.
  • the horizontal axis represents each of 370 olfactory receptors to be tested, and the vertical axis represents the relative response intensity of the receptors.
  • olfactory receptor OR1A1 Response of olfactory receptor OR1A1 expressing cells to various test substances.
  • the horizontal axis represents the test substance concentration, and the vertical axis represents the response intensity. Mock represents the response of cells that do not express olfactory receptors.
  • AB Responses of olfactory receptor OR10G4 (A) and OR9Q2 (B) expressing cells to various test substances.
  • the horizontal axis represents the test substance concentration, and the vertical axis represents the response intensity.
  • the bar represents the average value of the evaluation results of the sensory test, and the error bar represents standard error (SE).
  • olfactory receptor polypeptide refers to an olfactory receptor or a polypeptide having a function equivalent thereto
  • polypeptide having a function equivalent to an olfactory receptor refers to an olfactory receptor and Similarly, it can be expressed on the cell membrane, activated by the binding of odor molecules, and when activated, the amount of intracellular cAMP is activated by activating adenylate cyclase in combination with intracellular G ⁇ s or G ⁇ olf.
  • cent cross adaptation or olfactory cross adaptation relating to a target odor means that an odor of a substance other than the causative substance of the target odor is received in advance and used to the odor. It refers to a phenomenon in which the olfactory sensitivity to the causative agent of the target odor is reduced or changed.
  • smelling cross adaptation is a phenomenon based on olfactory receptor agonism.
  • an olfactory receptor for a target odor causative substance responds to a different odor causative substance prior to the response to the target odor causative substance, and then the response adapts, so that the target odor causative substance is later When exposed, only a low response is possible, resulting in a decrease or alteration in the intensity of the target odor recognized by the individual. Therefore, a substance that activates the response of the olfactory receptor to the causative substance of the target odor, such as an olfactory receptor agonist, can be used for the cross-adaptation of the target odor according to the present invention and the suppression of the target odor based on the target odor.
  • 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
  • amino acid sequences and nucleotide 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 present invention provides a method for selecting a pair of odorants that cause cross-adaptation using an olfactory receptor response as an index.
  • the present invention also provides a method for selecting a substance that suppresses the odor based on cross-adaptation using the activation of an olfactory receptor for an odor-causing substance as an index.
  • a substance capable of selectively deodorizing a target odor based on cross adaptation can be efficiently selected.
  • the inventors of the present invention have suggested that there is a high relationship between odor perception and specific olfactory receptor response, such as musk fragrance and olfactory receptor OR5AN1, and guaiacol (2-methoxyphenol) and olfactory receptor OR10G4.
  • olfactory receptor response such as musk fragrance and olfactory receptor OR5AN1
  • guaiacol (2-methoxyphenol) and olfactory receptor OR10G4 By using a simple odor-olfactory receptor model, we succeeded in verifying the involvement of the olfactory receptor level in odor cross-adaptation for the first time.
  • the present inventors have discovered the principle that another substance recognized by an olfactory receptor that recognizes the target odor causing substance with high sensitivity causes cross-adaptation of the target odor.
  • the present inventors confirmed that the principle is universal as a result of further experiments with another odor-olfactory receptor model (p-cresol and OR9Q2). From the above, the present inventors have found that cross-adaptation can be examined for various odors by screening olfactory receptors for odor substance responsiveness and constructing a further odor-olfactory receptor model.
  • the present invention provides a method for screening or identifying a substance that causes cross-adaptation of a target odor and a substance that suppresses the target odor by cross-adaptation.
  • the present invention is a method for selecting a substance that causes cross-adaptation of a target odor, (1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent; (2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
  • a method comprising:
  • the present invention also provides a method for selecting a substance that suppresses the target odor, (1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent; (2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that suppresses the odor of the target;
  • a method comprising:
  • the present invention also provides a method for selecting a substance that causes cross-adaptation of a target odor, (1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent; (2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
  • a method comprising:
  • the present invention also provides a method for selecting a substance that suppresses the target odor, (1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent; (2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that suppresses the target odor;
  • a method comprising:
  • the above-described method of the present invention can be applied to all olfactory receptor polypeptides responsible for smell transmission and all odors recognized by them.
  • the method of the present invention may be a method performed in vitro or ex vivo.
  • a target odor to cause or suppress cross-adaptation
  • at least one olfactory receptor polypeptide having responsiveness to a causative substance of the target odor is prepared.
  • At least one olfactory receptor polypeptide that responds to a target odor-causing agent is searched for a group of olfactory receptor polypeptides from which the odor-causing agent is targeted. It can be obtained by identifying what responds.
  • the “olfactory receptor for the target odor causative agent” to be identified does not have to be all olfactory receptors capable of accepting the target odor causative agent, and is a receptor that plays a major role in the recognition of the target odor. Any receptor that can respond to the body, in other words, the causative substance of the target odor from a relatively low concentration, or exhibits a relatively high response to the causative substance of a certain concentration of odor may be used.
  • the dependence of the cellular response in which the receptor is expressed on the odor causative substance concentration is obtained, and the 50% effective concentration (EC50) calculated from the result is expressed as “an olfactory receptor for the causative substance of the target odor”. May be identified.
  • the “olfactory receptor for the target odor causative agent” to be identified is a receptor having a higher binding ability such as a low dissociation constant If it is.
  • an olfactory receptor polypeptide having responsiveness to a target odor causative substance is a response by various olfactory receptor polypeptides according to the method described in Reference Examples 1 and 2, as described in Example 1 described later. Can be identified by screening olfactory receptor polypeptides that respond to the causative agent of the target odor.
  • the olfactory receptor polypeptide used in the method of the present invention may be any mammal-derived olfactory receptor polypeptide.
  • mammal-derived olfactory receptor polypeptides include primates such as humans, chimpanzees, or olfactory receptor polypeptides derived from rodents such as mice and rats. And 400 or more olfactory receptors possessed by humans and polypeptides having functions equivalent thereto.
  • Information on human, mouse and rat olfactory receptors can be found in GenBank [www. ncbi. nlm. nih. gov].
  • the population of olfactory receptor polypeptides to be searched may be derived from a single mammalian species, or may contain olfactory receptor polypeptides derived from two or more different mammalian species. Good.
  • the population of olfactory receptor polypeptides to be searched is human, mouse and rat olfactory receptors, and at least 80% identical in amino acid sequence and having functions equivalent to those of olfactory receptors. More preferably, any of human olfactory receptors and polypeptides that are at least 80% identical in amino acid sequence to them and have functions equivalent to those of olfactory receptors including.
  • the olfactory receptor polypeptide can be used in any form as long as it does not lose responsiveness to the target odor causing substance.
  • 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 genetically engineered to express the olfactory receptor polypeptide or a culture thereof; 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 human olfactory receptor polypeptides.
  • the recombinant cell can be prepared by transforming a cell using a vector incorporating a gene encoding an olfactory receptor polypeptide.
  • the olfactory receptor polypeptide can also be expressed by directly introducing a gene transcription product into a cell.
  • a gene encoding RTP receptor-transporting protein
  • 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 registered in GenBank as GI: 50234917.
  • the type of target odor is not particularly limited, and generally known bad odors or unpleasant odors (for example, body odor, habit odor, halitosis, fecal odor, urine odor, tobacco odor, mold odor, dry odor, rot odor, garbage Odors, sewage odors, exhaust odors, duct odors, exhaust gas odors, etc.) as well as odors from foods or fragrance materials, odors from other substances (eg cosmetics, pharmaceuticals, cleaning agents, daily necessities, etc.), etc. Includes all the odors.
  • bad odors or unpleasant odors for example, body odor, habit odor, halitosis, fecal odor, urine odor, tobacco odor, mold odor, dry odor, rot odor, garbage Odors, sewage odors, exhaust odors, duct odors, exhaust gas odors, etc.
  • odors from foods or fragrance materials eg cosmetics, pharmaceuticals, cleaning agents
  • the causative substance of the target odor may be any substance that acts on the olfactory receptor to perceive the target odor.
  • the causative substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, or a compound, a composition or a mixture. Also good.
  • the causative substance is a volatile substance.
  • causative agent examples include muscone, a causative agent of musk odor, guaiacol, a causative agent of tobacco odor, p-cresol, a causative agent of urine odor, 4-methyl-3- Hexenoic acid, 3-mercapto-3-methylhexanol, 3-hydroxy-3-methylhexanoic acid and 3-methyl-2-hexenoic acid, causative substances of odor, diosmine and 2-methyliso Borneol, skatole and indole that cause fecal odor or bad breath, nonanoic acid, hexanoic acid and isovaleric acid that cause body odor, volatile sulfur that is a cause of bad odor emitted from garbage, sewage or drainage Compounds, and butyl acrylate, pyrazine derivatives, furaneol, sotron and the like.
  • the causative substance of the target odor is added to the olfactory receptor polypeptide, and its response to the causative substance 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.
  • a method for measuring the response of the olfactory receptor for example, measurement of intracellular cAMP amount.
  • intracellular cAMP amount for example, when an 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. Therefore, the response of the olfactory receptor polypeptide to the causative substance can be measured by using the amount of intracellular cAMP after the causative substance is added as an index.
  • Examples of the method for measuring the amount of cAMP include an ELISA method and a reporter gene assay.
  • Another method for measuring the response of an olfactory receptor polypeptide includes a calcium imaging method.
  • Yet another method includes measurement by electrophysiological techniques. In electrophysiological measurement, for example, a cell (such as Xenopus oocyte) in which an 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 measurement using an electrode membrane potential fixation method or the like.
  • an olfactory receptor polypeptide that responds to the target odor-causing substance is identified. Evaluation of responsiveness can be performed by comparing the response of the olfactory receptor polypeptide (test group) to which the causative substance is added with the control group.
  • the control group includes the olfactory receptor polypeptide to which the causative substance at different concentrations was added, the olfactory receptor polypeptide to which the causative substance was not added, the olfactory receptor polypeptide to which a control substance was added, and the cause The olfactory receptor polypeptide before adding a substance, cells in which the olfactory receptor polypeptide is not expressed, and the like can be mentioned.
  • the control group includes other olfactory receptor polypeptides having no response or low response to the causative agent.
  • the olfactory receptor polypeptide is identified as responding to the target odor causative agent. For example, if the response of the olfactory receptor polypeptide in the test group is enhanced to 200% or more, more preferably 300% or more, and even more preferably 400% or more compared to the control group, the olfactory receptor is enhanced. Polypeptides are identified as responding to the target odor causative agent. Alternatively, if the response of the olfactory receptor polypeptide in the test group is statistically significantly enhanced as compared to the control group, the olfactory receptor polypeptide is responsive to the target odor causing substance. Identified as
  • the target odor causative substance may be added to the identified olfactory receptor polypeptide at a different concentration, and the response may be measured by the same procedure. If the response increases in a concentration-dependent manner of the causative agent, it can be confirmed that the olfactory receptor polypeptide is responsive to the causative agent of the target odor.
  • olfactory receptor polypeptides having responsiveness to a target odor causative agent When multiple types of olfactory receptor polypeptides having responsiveness to a target odor causative agent are found and their response intensities are different from each other, a relatively high responsiveness to the causative agent is shown among them 1
  • More than one species of olfactory receptor polypeptide can be further selected. For example, one, two, or three or more olfactory receptor polypeptides can be selected from the ones with the highest responsiveness to the target odor-causing substance, or the one with the highest responsiveness is selected. Can do. Alternatively, the olfactory receptor polypeptide having the lowest responsiveness to the target odor-causing substance can not be selected, or two or more of the responsiveness can be not selected.
  • the “selected” one or more olfactory receptor polypeptides are 200% or more, preferably 300% or more, more preferably 400, compared to each of the “non-selected” olfactory receptor polypeptides. % Responsiveness.
  • the sensitivity of the olfactory receptor polypeptide to the target odor causative substance can be evaluated using EC50, response threshold, etc. as an index, and a highly sensitive olfactory receptor polypeptide can be selected.
  • a highly sensitive and highly responsive olfactory receptor polypeptide can be selected based on the evaluation of responsiveness and response sensitivity.
  • Olfactory receptor polypeptides exhibit different basal activities depending on the type when expressed in cultured cells. Therefore, when comparing responses between olfactory receptor polypeptides, it is desirable to standardize the response values for each olfactory receptor polypeptide and compare the obtained normalized values between olfactory receptor polypeptides.
  • a standardization method for example, for a cell expressing one type of olfactory receptor, a relative value of a signal with respect to an odor stimulation when the signal value when no odor stimulation is performed is set to 1, 1 For example, a method of subtracting a signal value when no odor stimulation is performed from a signal value with respect to the odor stimulation for cells expressing various types of olfactory receptors.
  • an olfactory receptor polypeptide having responsiveness to the target odor-causing substance can be identified.
  • at least one olfactory receptor polypeptide that is responsive to the target odor-causing substance is prepared.
  • Examples of the at least one olfactory receptor polypeptide prepared in the method of the present invention include a human, mouse or rat olfactory receptor responsive to a causative agent of a target odor, and an amino acid sequence thereof. And at least one selected from the group consisting of polypeptides that are at least 80% identical and responsive to the target odor causative agent.
  • a human olfactory receptor that is responsive to a target odor causative agent, and at least 80% identical in amino acid sequence thereto, and responsive to the target odor causative agent include at least one selected from the group consisting of polypeptides.
  • Further preferred examples include a group of human olfactory receptors that are responsive to a target odor-causing substance, the olfactory receptors having the highest responsiveness, and at least 80% identical in amino acid sequence, and examples thereof include at least one selected from the group consisting of polypeptides having responsiveness to a target odor causing substance.
  • the most sensitive and highly responsive olfactory receptor and the amino acid sequence thereof are at least 80% identical.
  • the olfactory receptor polypeptide prepared in the method of the present invention may be at least one responsive to the target odor causative substance, or any two or more of them may be combined.
  • the target odor is a musk fragrance
  • the odor-causing substance is a musk fragrance
  • the olfactory receptor polypeptide used is represented by OR5AN1 (SEQ ID NO: 2) and SEQ ID NO: 2. And at least one selected from the group consisting of polypeptides having a responsiveness to musk fragrances (see FIG. 1).
  • the target odor is guaiacol (2-methoxyphenol)
  • the odor-causing substance is guaiacol
  • the olfactory receptor polypeptide used is OR10G4 (SEQ ID NO: 4)
  • Guaiacol is known to be a main causative substance of tobacco odor (Japanese Patent Laid-Open No. 2006-321944). Therefore, in one embodiment of the method of the present invention, the target odor is tobacco odor, and the olfactory receptor polypeptide used is represented by OR10G4 (SEQ ID NO: 4), which is a receptor for guaiacol, and SEQ ID NO: 4. And at least one selected from the group consisting of polypeptides that are responsive to tobacco odor-causing substances, preferably guaiacol.
  • the target odor is urine odor.
  • JP-A-2009-132770 discloses that p-cresol is the component that has the highest contribution to urine odor emitted from scattered and dried urine or diapers left after use, and p-cresol and other carbon numbers. It is described that a urine odor can be reproduced by a composition with 6 to 10 phenolic compounds.
  • International Publication No. 2009/037861 describes that when ⁇ -glucuronidase produced by bacteria acts on urine, p-cresol or other components increase in the urine, and the urine odor intensity is significantly increased. ing.
  • examples of the “urine odor-causing substance” in the present invention include urinary ⁇ -glucuronidase-treated product or an extract thereof, and p-cresol.
  • examples of the urinary ⁇ -glucuronidase-treated product include those obtained by adding ⁇ -glucuronidase to urine and urine on which ⁇ -glucuronidase produced by bacteria in the urine acts. These urine processed products or extracts thereof contain p-cresol.
  • OR9Q2 has been found as an olfactory receptor that recognizes p-cresol, which is a urinary odor causative substance (see FIGS. 1 and 2).
  • OR9Q2 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, which is registered as GI: 284413710 with GenBank. Therefore, preferably, when the target odor is urine odor in the method of the present invention, the olfactory receptor polypeptide used is at least 80% identical to OR9Q2 (SEQ ID NO: 6) and the amino acid sequence represented by SEQ ID NO: 6. It is at least one selected from the group consisting of an amino acid sequence and a polypeptide having responsiveness to a substance that causes urine odor, preferably p-cresol.
  • a test substance is added to the olfactory receptor polypeptide having responsiveness to the target odor-causing substance.
  • the test substance used in the method of the present invention is not particularly limited as long as it is a substance that is desired to be used as a substance that causes cross-adaptation of the target odor or as a substance that suppresses the target odor.
  • 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 test substance is different from the causative substance of the target odor.
  • the test substance is a volatile substance and has a smell different from the target odor.
  • the test substance is a fragrance having an odor different from the target odor.
  • the response of the olfactory receptor polypeptide to the test substance is measured.
  • the measurement may be performed according to the method described above with respect to the measurement of the response of the olfactory receptor polypeptide to the causative agent of the target odor.
  • the receptor activation effect of the test substance is evaluated, and the test substance causing cross adaptation of the target odor is identified.
  • the effect of the test substance can be evaluated by comparing the response of the olfactory receptor (test group) to which the test substance is added with the control group.
  • test 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.
  • the control group includes other olfactory receptor polypeptides that have no response or low response to the test substance.
  • the test substance can be evaluated as a substance that activates the response of the olfactory receptor polypeptide.
  • the effect of the test substance on the response of the olfactory receptor polypeptide is between the test substance addition group and the non-addition group, between the test substance addition group and the control substance addition group, before and after the addition of the test substance, or It can be evaluated by comparing the response of the olfactory receptor polypeptide between the olfactory receptor polypeptide-expressing cell group and the non-expressing cell group.
  • test substance When the response of the olfactory receptor polypeptide is induced by addition of a test substance, the test substance is evaluated as a substance that activates the response of the olfactory receptor polypeptide in response to the causative substance of the target odor.
  • the test is performed.
  • a substance can be evaluated as a substance that activates the response of an olfactory receptor polypeptide.
  • the response of the olfactory receptor polypeptide in the test substance-added group is statistically significantly enhanced as compared with the control group, the test substance activates the response of the olfactory receptor polypeptide. It can be evaluated as a substance.
  • the test substance that activates the response of the olfactory receptor polypeptide in response to the target odor-causing substance obtained above is selected as a substance that causes cross-adaptation of the target odor. That is, in the presence of the test substance, the olfactory receptor that recognizes the target odor is activated and subsequently responsiveness decreases due to adaptation, so that the responsiveness to the target odor causing substance added later is impaired. As a result, cross-adaptation of the target odor is caused by the test substance.
  • the test substance that activates the response of the olfactory receptor polypeptide that responds to the target odor causative substance obtained above is selected as a substance that suppresses the target odor. That is, the test substance is a substance that can suppress the target odor by causing cross adaptation of the target odor.
  • the test substance selected in the above procedure may be subjected to a sensory test to further evaluate its cross-adaptation ability or target odor suppression ability.
  • the sensory test can be performed in accordance with a deodorant evaluation procedure normally performed in the field, but preferably, the test substance and target for the evaluator are considered in consideration of the test substance being a cross-adaptation inducer.
  • the order of application of odor-causing substances is adjusted.
  • the evaluator first smells the candidate test substance selected in the above procedure and adapts to the smell.
  • the evaluator then smells the target and evaluates its intensity.
  • the evaluation results obtained are compared with the intensity of the target odor when not adapted to the test substance.
  • the test substance evaluated as having reduced the intensity of the target odor is selected as a substance that causes cross adaptation to the target odor or a substance that suppresses the target odor.
  • the substance selected by the method of the present invention is a substance that can suppress the target odor based on cross adaptation.
  • One embodiment of using a substance that suppresses the target odor obtained by the method of the present invention is as follows: First, the target is targeted to a subject who desires suppression of the target odor. Before being exposed to the odor of the target, the odor of the substance that suppresses the target odor is smelled. Alternatively, a substance that suppresses the odor of the target is applied to the subject so that the odor is stronger than the odor of the target. As a result, even if the subject is exposed to the target odor, the odor sensitivity to the target odor is reduced, and therefore the target odor is felt weak or no longer felt.
  • Examples of the application of the substance that suppresses the target odor obtained in the present invention include placing the substance in front of or in a toilet; carrying the substance to a person involved in excretion treatment in a ward or a nursing facility Or a method of exposing the substance before the treatment; a paper diaper or a sanitary product containing the substance; an undergarment, underwear, linen or other clothing containing the substance, a cloth product, or a fabric; Laundry detergents or softeners containing substances; cosmetics, detergents, deodorants and other external preparations containing the substances, pharmaceuticals, foods, etc .; production lines for products with a target odor or target odors generated Examples include, but are not limited to, application to the environment.
  • a method for selecting a substance that causes cross-adaptation of a target odor (1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent; (2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor; Including the method.
  • a method for selecting a substance that suppresses a target odor (1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent; (2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that suppresses the odor of the target; Including the method.
  • the olfactory receptor polypeptide searched in (1) above is at least 80% identical in amino acid sequence to human, mouse and rat olfactory receptors and equivalent to the olfactory receptors ⁇ 1> or ⁇ 2>
  • a method for selecting a substance that causes cross-adaptation of a target odor (1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent; (2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor; Including the method.
  • a method for selecting a substance that suppresses a target odor (1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent; (2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response; (3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that suppresses the target odor; Including the method.
  • the at least one olfactory receptor polypeptide is preferably at least one selected from the group consisting of: Rat or mouse olfactory receptor; Human olfactory receptor; and 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more in amino acid sequence with the rat, mouse or human olfactory receptor More preferably, the polypeptide is 99% or more identical and responsive to the target odor-causing substance.
  • the olfactory receptor polypeptide is expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide, any of ⁇ 1> to ⁇ 6> The method according to claim 1.
  • the response of the olfactory receptor polypeptide is measured by intracellular cAMP amount measurement by ELISA or reporter gene assay, or by calcium imaging or electrophysiological technique, any one of ⁇ 1> to ⁇ 7> The method according to claim 1.
  • ⁇ 9> Preferably, (4) a substance that causes cross adaptation to the target odor or a substance that suppresses the target odor among the substances selected in (3) above by a sensory test.
  • a substance that causes cross adaptation to the target odor or a substance that suppresses the target odor among the substances selected in (3) above by a sensory test Preferably, (4) a substance that causes cross adaptation to the target odor or a substance that suppresses the target odor among the substances selected in (3) above by a sensory test.
  • ⁇ 10> The method according to any one of ⁇ 1> to ⁇ 9>, wherein the target odor is preferably a tobacco odor or a urine odor.
  • the method according to any one of ⁇ 4> to ⁇ 9> preferably,
  • the target odor is tobacco odor
  • the at least one olfactory receptor polypeptide is: A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4; and 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably, with the amino acid sequence represented by SEQ ID NO: 4 Is a polypeptide that is 98% or more, preferably 99% or more identical and responsive to tobacco odor-causing substances,
  • the target odor is urine odor
  • the at least one olfactory receptor polypeptide is: A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6; and 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably, with the amino acid sequence represented by SEQ ID NO: 6 Is a polypeptide which is 98% or more, preferably 99% or more, and has a response to a urine odor causing substance,
  • test substance is preferably a substance different from the target odor causing substance.
  • ⁇ 14> The method according to any one of ⁇ 1> to ⁇ 13>, preferably, among at least one olfactory receptor polypeptide exhibiting responsiveness to the target odor-causing substance.
  • the odorant receptor polypeptide that responds to the causative agent of the target odor is further selected.
  • Reference Example 1 Preparation of cells expressing human olfactory receptor 1
  • Cloning of human olfactory receptor gene 370 human olfactory receptors listed in Tables 1-1 and 1-2 based on sequence information registered in GenBank And genes encoding human olfactory receptors OR5AN1, OR10G4, and OR9Q2 (SEQ ID NOs: 1, 3, and 5 respectively) were cloned. Each gene was cloned by PCR using human genomic DNA female (G1521: Promega) as 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.
  • Example 3 Production of olfactory receptor-expressing cells
  • HEK293 cells each expressing 370 types of human olfactory receptors were produced.
  • 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 4.4 ⁇ L was added to each well of a 384 well plate (BioCoat).
  • 40 ⁇ L of HEK293 cells (20 ⁇ 10 4 cells / cm 2 ) were seeded in each well and cultured for 24 hours in an incubator maintained at 37 ° C. and 5% CO 2 .
  • Example 1 2 HEK293 cells expressing human olfactory receptors OR5AN1, OR10G4, OR9Q2 or OR1A1 were prepared.
  • a reaction solution having the composition shown in Table 3 was prepared and allowed to stand in a clean bench for 15 minutes, and then 10 ⁇ L was added to each well of a 96-well plate (BioCoat).
  • 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 in the same manner.
  • Luciferase Assay The olfactory receptor expressed in HEK293 cells is conjugated 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 downstream of the CMV promoter hRluc-CMV was simultaneously introduced, and used as an internal standard for correcting errors in gene transfer efficiency or cell number.
  • 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 induced by odor substance stimulation by fLuc / hRluc in a cell not subjected to odor substance stimulation was calculated as a fold increase and used as an index of response intensity. Dose response curves were analyzed using GraphPad Prism.
  • Example 1 Response of olfactory receptor to odorant 1) Identification of olfactory receptor responding to odorant The medium was removed from the culture of olfactory receptor-expressing cells prepared according to Reference Example 1, and the odorant (Muscone, Guiacol or p-) Cresol) was added. 30 ⁇ L of Muscone or Guaiacol-containing DMEM medium (Nacalai) was added to each well of the 384-well plate containing the culture (final concentrations: Muscone 100 ⁇ M, Guaicol 1 mM). In addition, 75 ⁇ L of CD293 medium (Invitrogen) containing p-Cresol was added to each well of a 96-well plate containing the culture (final concentration 1 mM).
  • the luciferase assay is performed by the method of Reference Example 2 to determine the response intensity of the olfactory receptor to the odorant ( fold increase) was measured.
  • the results are shown in FIG.
  • the vertical axis represents the relative response intensity of each receptor-expressing cell to the odor stimulation when the response intensity under the condition without odor stimulation is 1.
  • OR5AN1, OR10G4, and OR9Q2 were identified as receptors that gave the highest responsiveness to Muscone, Guiacol, and p-Cresol, respectively.
  • OR9Q2 Odor substance concentration dependence of olfactory receptor response According to the methods described in Reference Examples 1 and 2, the response of OR9Q2 to different concentrations of p-Cresol was measured. As a result, OR9Q2 showed a p-cresol concentration-dependent response and was confirmed to be a p-Cresol receptor (FIG. 2).
  • Example 2 Action of odorant substance on olfactory receptor 1 Measurement of olfactory receptor response The medium was removed from the culture of olfactory receptor-expressing cells prepared according to Reference Example 1, and from 0.3 ⁇ M in DMEM medium (Nacalai Tesque). 75 ⁇ L of a solution containing an odorant prepared to 100 ⁇ M was added. After culturing the cells in a CO 2 incubator for 3 to 4 hours to fully express the luciferase gene in the cells, the luciferase assay is performed by the method of Reference Example 2, and the response intensity of the olfactory receptor to the odorant (fold increase) ) was measured.
  • OR5AN1 is an olfactory receptor that responds to musk fragrances.
  • OR5AN1 responded to these substances in a concentration-dependent manner (FIG. 3). These responses were dependent on OR5AN1, since they were not observed in cells that do not express OR5AN1 (Mock).
  • Ambrettolide, Phenyl ethyl alcohol (PEA), Lyral®, and Cis-3-hexanol did not cause an OR5AN1 response.
  • OR1A1 (FIG. 1) showing weaker responsiveness to Muscone than OR5AN1 was also activated by PEA and Cis-3-hexanol (FIG. 4). It was confirmed that OR10G4 has responsiveness to Guiacol and Ethylvanillin (FIG. 5A).
  • OR9Q2 as shown in FIG. 2, responsiveness to p-Cresol, which is a urine odor-causing substance, was confirmed, and responsiveness to p-Creyl Acetate, p-Creyl iso butyrate, and p-Methyl quinoline was further improved. Was found (FIG. 5B). On the other hand, it was found that neither OR10G4 nor OR9Q2 has responsiveness to Muscone (FIG. 5C).
  • Example 3 Sensory Evaluation of Cross Adaptation Action by Odor Substance 1
  • a target odor substance was added to a glass bottle (Saiyo Glass No. 11, volume 110 mL) to obtain a target sample.
  • Muscone As the target odor substance, Muscone, Guiacol or p-Cresol was used.
  • the Muscone used was a cotton ball soaked with 1 mg.
  • 1 mL of 10 ppm mineral oil (Sigma-Aldrich) solution was used.
  • a test substance was added to a glass bottle to obtain a test sample.
  • Ethylvanillin powder As a test substance, 5 mg of Ethylvanillin powder, or 1 mL of p-Cresyl Acetate, p-Creyl isobutyrate, or p-Methyl quinoline mineral oil (Sigma-Aldrich) solution prepared to 1000 ppm was used.
  • the sensory test was conducted in a single blind manner by 5 to 8 panelists. The panelist was first presented with the target sample and the odor intensity was answered. The test samples were then presented for 2 minutes or until no odor was detectable. Then, the target sample was again presented and the odor intensity was evaluated. This was set as 1 set, and 4 sets per person per day were set as the upper limit. There was a break of at least 10 minutes between each set.
  • the intensity of the target odor evaluated was entered on a straight line of 9.5 cm showing the scales of “No Odor” and “Strong Odor” at both ends, respectively.
  • the distance (A) between the target odor intensity entry position and No Odor in the first evaluation and the distance (B) between the target odor intensity entry position and No Odor in the second evaluation are obtained, and the% odor with respect to the first evaluation Intensity (% Oor intensity of initial estimate) [B / A * 100-100] was calculated.
  • the lower this value is, the stronger the intensity of the target odor smelled at the second time is compared with the first time, that is, the stronger the cross adaptation. If this value is ⁇ 100%, it means that the target odor was not felt at all for the second time.
  • OR10G4 is the olfactory receptor having the highest response to the odor of Guiacol (FIG. 1).
  • the odor of Guiacol was cross-adapted by Ethylvanillin that activates OR10G4 (FIG. 6B).
  • the odor of Guiacol was not cross-adapted by Muscone that did not activate OR10G4, and the smell of Ethylvanillin was not cross-adapted by Muscone. From this result, it was shown that the principle that another substance recognized by the olfactory receptor highly reactive to the target odor causative substance causes cross-adaptation of the target odor can also be applied to OR10G4.
  • Muscone that does not activate OR9Q2 does not cause cross adaptation to the odor of p-Cresol, nor to the odor of p-Cresyl Acetate, p-Cresyl iso butyrate, and p-Methyl quinoline. It was.
  • the present invention provides a novel method for identifying an odorant capable of suppressing a target odor based on cross-adaptation, using activation of an olfactory receptor response as an index.

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Abstract

Provided is a method for searching for a substance that controls a target odor by cross adaptation. The method for selecting a substance that induces cross adaptation to a target odor comprises: searching olfactory receptor polypeptides and identifying one that responds to the target odor causative substance; adding a test substance that is different from the target odor causative substance and measuring the response; and selecting the test substance that has activated the response of the olfactory receptor polypeptide as the substance that induces cross adaptation to the target odor.

Description

匂い抑制物質の選択方法How to select odor control substances
 本発明は、匂いの順応機構に基づいて匂いを抑制する方法に関する。 The present invention relates to a method for suppressing odor based on an odor adaptation mechanism.
 ヒト等の哺乳動物においては、匂い物質は、鼻腔内最深部に広がる嗅上皮に存在する嗅神経細胞の嗅覚受容体によって認識される。鼻腔内に取り込まれた匂い分子は、嗅覚受容体に作用して活性化させ、活性化した嗅覚受容体に引き起こされた嗅神経細胞からのシグナルが中枢神経系へと伝達されることにより、匂いが知覚される。嗅覚受容体をコードする遺伝子はヒトの場合400種以上存在することが予想されている。特定の匂い物質に対して我々が知覚する匂いの質は、該匂い物質によって上記400種以上の嗅覚受容体のうちのどの組み合わせが活性化されたかによって決定されると考えられている。 In mammals such as humans, odorous substances are recognized by olfactory receptors of olfactory neurons existing in the olfactory epithelium that spreads in the deepest part of the nasal cavity. The odor molecules taken into the nasal cavity act on the olfactory receptor to activate it, and the signal from the olfactory nerve cell caused by the activated olfactory receptor is transmitted to the central nervous system. Is perceived. In humans, it is expected that more than 400 genes encoding olfactory receptors exist. It is believed that the odor quality we perceive for a particular odorant is determined by which combination of the 400 or more olfactory receptors activated by the odorant.
 匂いとその認識に関わる嗅覚受容体との関係については、既に様々な報告がある。例えば、2014年マウスを使った研究により、数十万ともいわれる匂い物質の中でもムスク系の香料は、例外的に極めて少数の嗅覚受容体で認識されることが示唆された。ヒトでも、ムスクを認識する嗅覚受容体としてはOR5AN1の1種類のみが見出されており、この1種類の受容体がムスクの匂い認識に大きく貢献していることが示唆されている(非特許文献1)。また例えば、グアイアコール(2-メトキシフェノール)という匂い物質の認識には、OR10G4という1種類の嗅覚受容体が高く寄与していることが示唆されている(非特許文献2)。 There are already various reports on the relationship between odors and olfactory receptors involved in their recognition. For example, a study using mice in 2014 suggested that musk-based fragrances are exceptionally recognized by very few olfactory receptors among hundreds of thousands of odorants. Even in humans, only one type of OR5AN1 has been found as an olfactory receptor that recognizes musk, and it has been suggested that this one type of receptor greatly contributes to musk odor recognition (non-patented). Reference 1). In addition, for example, it has been suggested that one type of olfactory receptor called OR10G4 contributes to recognition of an odorous substance called guaiacol (2-methoxyphenol) (Non-patent Document 2).
 衣類など繊維製品から生じる「生乾き臭」、頭部、口腔、鼠蹊部、足の裏などから発生する「体臭」、排泄されてから長時間が経過した尿から発せられる「尿臭」など、様々な悪臭が、近年の清潔志向の高まりに伴い問題となっている。これら悪臭の原因となる匂い物質の多くは、分子量約30~300の揮発性の低分子有機化合物である。悪臭に対する不快感を軽減する方法としては、中和反応を利用した化学消臭、悪臭物質を多孔質表面などに吸着させる物理消臭、別の匂い物質を使った感覚消臭がある。さらに感覚消臭としては、(1)他の匂い物質を悪臭の環境に導入し、悪臭と一緒に嗅がせる方法(マスキング)、(2)悪臭を嗅ぐ前に、他の匂い物質を嗅がせることによって、悪臭に対する人間の嗅覚を低下させる方法(交差順応)がある。 "Dry odor" generated from textiles such as clothing, "Body odor" generated from the head, oral cavity, buttocks, soles, etc., "Urine odor" generated from urine that has been excreted for a long time, etc. The bad odor has become a problem with the recent increase in cleanliness. Many of these odorous substances that cause malodor are volatile low-molecular organic compounds having a molecular weight of about 30 to 300. As a method for reducing unpleasant odor, there are chemical deodorization using a neutralization reaction, physical deodorization by adsorbing a malodorous substance on a porous surface, and sensory deodorization using another odorous substance. In addition, sensory deodorization includes (1) the method of introducing other odorous substances into the malodorous environment and smelling them with the bad odor (masking), and (2) smelling other odorous substances before smelling the bad odor. There is a method (cross-adaptation) that reduces the human sense of smell against bad odors.
 近年、感覚消臭を引き起こす嗅覚メカニズムを解明し、それに基づいて効率的に感覚消臭技術を開発する取り組みがなされている。例えば、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が報告されている。この事実を踏まえて、マスキングによる感覚消臭(1)の一種として、悪臭を認識する嗅覚受容体のアンタゴニストを利用した技術が報告されている(特許文献1)。このアンタゴニストを利用した技術は、嗅覚受容体を直接制御するという観点からは、現在のところ唯一の感覚消臭技術である。 In recent years, efforts have been made to elucidate the olfactory mechanism that causes sensory deodorization and to develop sensory deodorization technology efficiently based on that mechanism. For example, it has been reported that an odor molecule that activates one olfactory receptor acts as an antagonist that inhibits activation of another olfactory receptor. Based on this fact, a technique using an olfactory receptor antagonist that recognizes malodor has been reported as a kind of sensory deodorization (1) by masking (Patent Document 1). The technology using this antagonist is currently the only sensory deodorization technology from the viewpoint of directly controlling olfactory receptors.
 感覚消臭(2)の方法は、嗅覚の交差順応という生理現象に基づいた方法である。嗅覚の交差順応とは、ある一つの匂い物質に慣れることで、異なる匂い物質にも慣れが生じ嗅覚感度が低下することと定義される(非特許文献3)。すなわち、悪臭ではない匂いに慣れさせることで、悪臭に対しても慣れが生じてその知覚が抑制される。特許文献2では、腋臭の原因成分に化学構造が酷似するがより弱い又は不快でない匂いを有する物質を嗅がせ続けることによって、該腋臭原因成分に対する嗅覚感度を低下させる、交差順応に基づく匂い抑制方法が記載されている。また特許文献3には、天然フレーバーと、その匂いを模したイミテーションフレーバーとの間の匂い類似性を、交差順応に基づいて評価する方法が記載されている。特許文献3では、二つのフレーバー組成物が同質の匂いであるほど強い交差順応が生じ、その順応の影響が脳血流量変化に表れると想定して、脳血流量変化を指標とすることで二つのフレーバーの類似性を評価している。 The method of sensory deodorization (2) is a method based on the physiological phenomenon of cross adaptation of olfaction. The cross-adaptation of olfaction is defined as habituation to a different odor substance due to habituation to a certain odor substance and a decrease in olfactory sensitivity (Non-patent Document 3). That is, by getting used to odors that are not offensive odor, habituation occurs even for bad odors, and perception is suppressed. In Patent Document 2, an odor suppression method based on cross-adaptation that reduces the olfactory sensitivity to the odor-causing component by continuing to smell a substance that has a chemical structure that closely resembles the odor-causing component but has a weaker or unpleasant odor Is described. Patent Document 3 describes a method for evaluating the odor similarity between a natural flavor and an imitation flavor simulating the odor based on cross adaptation. In Patent Document 3, assuming that two flavor compositions have the same odor, stronger cross adaptation occurs, and the effect of the adaptation appears in cerebral blood flow change. Evaluates the similarity of two flavors.
 交差順応による匂い感受性の低下は、嗅覚受容体レベルのみならず嗅神経細胞レベル、神経回路レベルなど多様な機序で起こり得る。嗅覚の交差順応のメカニズムとして様々な仮説が考えられてきた。一つは、交差順応は、高次脳領域の神経細胞で起こるという仮説である。すなわち、末梢の嗅神経細胞からの情報が統合され匂いの質が解読される高次脳領域の神経細胞が、変化せずに留まる匂い情報に不要に応答し続けないよう感受性を失うメカニズムである。もう一つは、交差順応は、末梢の嗅神経細胞の嗅覚受容体のレベルで起こるという仮説である。すなわち、ある匂い物質の嗅覚受容体が、予め別の物質に応答した結果脱感作して、後から曝露された当該匂い物質に対してシグナルを流さなくなるというメカニズムが推量される。前者の仮説については、匂いの情報がどのような高次脳神経回路で処理されているかが未だ不明であるため、検証が困難である。一方、後者の仮説も、400種以上といわれる嗅覚受容体の数の多さと、匂い物質の認識におけるそれらの組み合わせの複雑さのために、やはり検証が困難であった。 The decrease in odor sensitivity due to cross-adaptation can occur by various mechanisms such as olfactory receptor level, neural circuit level as well as olfactory receptor level. Various hypotheses have been considered as the mechanism of olfactory cross-adaptation. One is the hypothesis that cross-adaptation occurs in neurons in higher brain regions. In other words, it is a mechanism that loses sensitivity so that neurons in the higher brain region where information from peripheral olfactory neurons is integrated and odor quality is decoded do not continue to respond unnecessarily to odor information that remains unchanged . Another is the hypothesis that cross-adaptation occurs at the level of olfactory receptors in peripheral olfactory neurons. That is, a mechanism is presumed in which an olfactory receptor of a certain odor substance desensitizes as a result of responding to another substance in advance and does not send a signal to the odor substance exposed later. The former hypothesis is difficult to verify because it is still unclear what kind of higher-order brain circuit processes odor information. On the other hand, the latter hypothesis was still difficult to verify due to the large number of olfactory receptors, said to be more than 400, and the complexity of their combination in odorant recognition.
 また従来、ある匂いの交差順応を引き起こす物質については、その匂いの原因物質と化学構造が酷似しているか、若しくは匂いの質が似た物質であるとの推定がなされている(非特許文献3)。一方で、そのような原則はいずれも成立しないという指摘もなされている(非特許文献4)。非特許文献5では、ある一つのラット嗅覚受容体に受容された匂い物質同士が、良く似た匂いの質を呈し、それら匂い物質はヒトに交差順応を起こしたことが報告されている。しかし、一般的に一つの匂い物質は、選択性の異なる多数の嗅覚受容体で認識される。 Conventionally, it has been estimated that a substance causing cross-adaptation of a certain odor has a chemical structure very similar to that of the odor causing substance or a substance having a similar odor quality (Non-patent Document 3). ). On the other hand, it has also been pointed out that none of these principles holds (Non-Patent Document 4). In Non-Patent Document 5, it is reported that odor substances received by a single rat olfactory receptor have similar odor qualities, and these odor substances cause cross-adaptation in humans. However, in general, one odorant is recognized by many olfactory receptors with different selectivity.
 このように、交差順応が生じる生体メカニズムは未だ解明されておらず、また交差順応を引き起こす匂い物質を同定する方法も確立されていない。したがって、従来、交差順応を人為的かつ計画的に引き起こすことによって匂いを抑制する技術の開発は、極めて困難であった。 Thus, the biological mechanism in which cross-adaptation occurs has not yet been elucidated, and a method for identifying an odor substance that causes cross-adaptation has not been established. Therefore, it has been extremely difficult to develop a technique for suppressing odor by causing cross adaptation to occur artificially and systematically.
(特許文献1)特許第5646255号公報
(特許文献2)特開2005-53887号公報
(特許文献3)特許第4966790号公報
(非特許文献1)Shirasu M. et al., Neuron, 81:165-178, 2014
(非特許文献2)Mainland J.D. et al., Nat. Neurosci., 17(1):114-20, 2014
(非特許文献3)川崎通昭・堀内哲嗣郎「嗅覚とにおい物質」p71-72、社団法人におい・かおり環境協会、1998年
(非特許文献4)Pierce J.D., Chemical senses, 21:223-237, 1996
(非特許文献5)Chemosensory Perception, 3(3):149-155, 2010
(Patent Literature 1) Japanese Patent No. 5646255 (Patent Literature 2) Japanese Patent Laid-Open No. 2005-53887 (Patent Literature 3) Japanese Patent No. 4966790 (Non-Patent Literature 1) Shirasu M. et al., Neuron, 81: 165 -178, 2014
(Non-Patent Document 2) Mainland JD et al., Nat. Neurosci., 17 (1): 114-20, 2014
(Non-patent document 3) Kawasaki Michiaki and Horiuchi Tetsuro “Smell and smell substance” p71-72, Association of smell and smell, 1998 (Non-patent document 4) Pierce JD, Chemical senses, 21: 223-237 , 1996
(Non-Patent Document 5) Chemosensory Perception, 3 (3): 149-155, 2010
 本発明は、標的の匂いの交差順応を引き起こす物質の選択方法であって、
(1)嗅覚受容体ポリペプチドを探索し、標的の匂いの原因物質に応答するものを同定すること、
(2)該同定された嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該嗅覚受容体ポリペプチドの応答を活性化させた該試験物質を、該標的の匂いの交差順応を引き起こす物質として選択すること、
を含む方法を提供する。
The present invention is a method for selecting a substance that causes cross-adaptation of a target odor,
(1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent;
(2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
A method comprising:
 また本発明は、標的の匂いを抑制する物質の選択方法であって、
(1)嗅覚受容体ポリペプチドを探索し、標的の匂いの原因物質に応答するものを同定すること、
(2)該同定された嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該嗅覚受容体ポリペプチドの応答を活性化させた該試験物質を、該標的の匂いを抑制する物質として選択すること、
を含む方法を提供する。
The present invention also provides a method for selecting a substance that suppresses the target odor,
(1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent;
(2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that suppresses the odor of the target;
A method comprising:
 さらに本発明は、標的の匂いの交差順応を引き起こす物質の選択方法であって、
(1)標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドを準備すること、
(2)該少なくとも1種の嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該少なくとも1種の嗅覚受容体ポリペプチドのいずれかの応答を活性化させた該試験物質を、該標的の匂いの交差順応を引き起こす物質として選択すること、
を含む方法を提供する。
Furthermore, the present invention provides a method for selecting a substance that causes cross-adaptation of a target odor,
(1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent;
(2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
A method comprising:
 さらに本発明は、標的の匂いを抑制する物質の選択方法であって、
(1)標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドを準備すること、
(2)該少なくとも1種の嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該少なくとも1種の嗅覚受容体ポリペプチドのいずれかの応答を活性化させた該試験物質を、該標的の匂いを抑制する物質として選択すること、
を含む方法を提供する。
Furthermore, the present invention is a method for selecting a substance that suppresses the odor of a target,
(1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent;
(2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that suppresses the target odor;
A method comprising:
ムスコン、グアイアコール、p-クレゾールに対する嗅覚受容体の応答。横軸は被験対象とした370種の各嗅覚受容体、縦軸は受容体の相対応答強度を表す。Olfactory receptor response to muscone, guaiacol, p-cresol. The horizontal axis represents each of 370 olfactory receptors to be tested, and the vertical axis represents the relative response intensity of the receptors. p-クレゾールに対する嗅覚受容体OR9Q2の応答。Mockは嗅覚受容体を発現させない細胞の応答を表す。エラーバー=±SE、n=3~4。Response of olfactory receptor OR9Q2 to p-cresol. Mock represents the response of cells that do not express olfactory receptors. Error bar = ± SE, n = 3-4. 嗅覚受容体OR5AN1発現細胞の各種試験物質に対する応答。横軸は試験物質濃度、縦軸は応答強度を示す。Mockは嗅覚受容体を発現させない細胞の応答を表す。エラーバー=±SE、n=3~4。Response of olfactory receptor OR5AN1 expressing cells to various test substances. The horizontal axis represents the test substance concentration, and the vertical axis represents the response intensity. Mock represents the response of cells that do not express olfactory receptors. Error bar = ± SE, n = 3-4. 嗅覚受容体OR1A1発現細胞の各種試験物質に対する応答。横軸は試験物質濃度、縦軸は応答強度を示す。Mockは嗅覚受容体を発現させない細胞の応答を表す。エラーバー=±SE、n=3。Response of olfactory receptor OR1A1 expressing cells to various test substances. The horizontal axis represents the test substance concentration, and the vertical axis represents the response intensity. Mock represents the response of cells that do not express olfactory receptors. Error bar = ± SE, n = 3. A~B:嗅覚受容体OR10G4(A)及びOR9Q2(B)発現細胞の各種試験物質に対する応答。横軸は試験物質濃度、縦軸は応答強度を示す。エラーバー=±SE、n=3。C:嗅覚受容体発現細胞のムスコンに対する応答。エラーバー=±SD、n=2。AB: Responses of olfactory receptor OR10G4 (A) and OR9Q2 (B) expressing cells to various test substances. The horizontal axis represents the test substance concentration, and the vertical axis represents the response intensity. Error bar = ± SE, n = 3. C: Response of olfactory receptor-expressing cells to muscone. Error bar = ± SD, n = 2. A:試験物質によるムスコンの匂いの交差順応(n=5~7)。B:試験物質によるグアイアコールの匂いの交差順応(n=7)。C:試験物質によるp-クレゾールの匂いの交差順応(n=8)。いずれも、バーは官能試験の評価結果の平均値、エラーバーは標準誤差(SE)を示す。A: Cross adaptation of Muscon odor by test substance (n = 5-7). B: Cross-adaptation of guaiacol odor by test substance (n = 7). C: Cross-adaptation of p-cresol odor by test substance (n = 8). In both cases, the bar represents the average value of the evaluation results of the sensory test, and the error bar represents standard error (SE).
発明の詳細な説明Detailed Description of the Invention
 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若しくはGαolf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。 In the present specification, “olfactory receptor polypeptide” refers to an olfactory receptor or a polypeptide having a function equivalent thereto, and “polypeptide having a function equivalent to an olfactory receptor” refers to an olfactory receptor and Similarly, it can be expressed on the cell membrane, activated by the binding of odor molecules, and when activated, the amount of intracellular cAMP is activated by activating adenylate cyclase in combination with intracellular Gαs or Gαolf. A polypeptide having a function of increasing
 本明細書において、標的匂いに関する「匂いの交差順応(又は嗅覚の交差順応)」とは、当該標的匂いの原因物質とは別の物質の匂いを予め受容し、その匂いに慣れることによって、該標的匂いの原因物質に対する嗅覚感受性が低下又は変化する現象を指す。本発明者らは、「匂いの交差順応」が、嗅覚受容体アゴニズムに基づく現象であることを明らかにした。すなわち、標的匂いの原因物質に対する嗅覚受容体が、該標的匂いの原因物質への応答に先だって異なる匂いの原因物質に応答し、その後応答が順応することにより、後から該標的匂いの原因物質に曝されても低い応答しかできず、その結果、個体に認識される標的匂いの強度の低下又は変質が生じる。したがって、本発明による標的匂いの交差順応、及びそれに基づく標的匂いの抑制には、嗅覚受容体アゴニスト等の、標的匂いの原因物質に対する嗅覚受容体の応答を活性化させる物質が使用され得る。 In the present specification, “scent cross adaptation (or olfactory cross adaptation)” relating to a target odor means that an odor of a substance other than the causative substance of the target odor is received in advance and used to the odor. It refers to a phenomenon in which the olfactory sensitivity to the causative agent of the target odor is reduced or changed. The present inventors have clarified that “smelling cross adaptation” is a phenomenon based on olfactory receptor agonism. That is, an olfactory receptor for a target odor causative substance responds to a different odor causative substance prior to the response to the target odor causative substance, and then the response adapts, so that the target odor causative substance is later When exposed, only a low response is possible, resulting in a decrease or alteration in the intensity of the target odor recognized by the individual. Therefore, a substance that activates the response of the olfactory receptor to the causative substance of the target odor, such as an olfactory receptor agonist, can be used for the cross-adaptation of the target odor according to the present invention and the suppression of the target odor based on the target odor.
 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、アミノ酸配列及びヌクレオチド配列に関する「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 amino acid sequences and nucleotide 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.
 本発明は、嗅覚受容体の応答を指標として交差順応を起こす匂い物質ペアを選択する方法を提供する。また本発明は、ある匂いの原因物質に対する嗅覚受容体の活性化を指標として、交差順応に基づいて該匂いを抑制する物質を選択する方法を提供する。 The present invention provides a method for selecting a pair of odorants that cause cross-adaptation using an olfactory receptor response as an index. The present invention also provides a method for selecting a substance that suppresses the odor based on cross-adaptation using the activation of an olfactory receptor for an odor-causing substance as an index.
 本発明によれば、交差順応に基づいて標的の匂いを選択的に消臭することができる物質を、効率良く選択することができる。 According to the present invention, a substance capable of selectively deodorizing a target odor based on cross adaptation can be efficiently selected.
 標的匂いの原因物質の認識に関わる嗅覚受容体の活動に着目することで、交差順応物質を探索できるという可能性があった。しかし、一般的に一つの匂い物質は複数の嗅覚受容体で認識されるため、そのような探索は複雑であるうえ、探索された物質が実際に標的匂いの交差順応を引き起こすかどうかも不明であった。 There was a possibility that cross-adapted substances could be searched by focusing on the activity of olfactory receptors involved in the recognition of the causative substances of the target odor. However, since one odorant is generally recognized by multiple olfactory receptors, such a search is complicated and it is not clear whether the searched substance actually causes cross-adaptation of the target odor. there were.
 本発明者らは、ムスク系香料と嗅覚受容体OR5AN1、及びグアイアコール(2-メトキシフェノール)と嗅覚受容体OR10G4という、匂い知覚と特定の嗅覚受容体応答との高い関連性が示唆されている単純な匂い-嗅覚受容体モデルを利用することで、匂いの交差順応への嗅覚受容体レベルでの関与を初めて検証することに成功した。その結果、本発明者らは、標的匂いの原因物質を高感度に認識する嗅覚受容体が認識する別の物質は、該標的匂いの交差順応を起こすという原則を発見した。さらに本発明者らは、別の匂い-嗅覚受容体モデル(p-クレゾールとOR9Q2)でのさらなる実験の結果、当該原則に普遍性があることを確認した。以上のことから本発明者らは、匂い物質応答性について嗅覚受容体をスクリーニングしてさらなる匂い-嗅覚受容体モデルを構築することで、種々の匂いについて交差順応を調べることができることを見出した。 The inventors of the present invention have suggested that there is a high relationship between odor perception and specific olfactory receptor response, such as musk fragrance and olfactory receptor OR5AN1, and guaiacol (2-methoxyphenol) and olfactory receptor OR10G4. By using a simple odor-olfactory receptor model, we succeeded in verifying the involvement of the olfactory receptor level in odor cross-adaptation for the first time. As a result, the present inventors have discovered the principle that another substance recognized by an olfactory receptor that recognizes the target odor causing substance with high sensitivity causes cross-adaptation of the target odor. Furthermore, the present inventors confirmed that the principle is universal as a result of further experiments with another odor-olfactory receptor model (p-cresol and OR9Q2). From the above, the present inventors have found that cross-adaptation can be examined for various odors by screening olfactory receptors for odor substance responsiveness and constructing a further odor-olfactory receptor model.
 上記原則に基づいて、本発明は、標的匂いの交差順応を引き起こす物質、及び交差順応により標的匂いを抑制する物質を、スクリーニング又は同定する方法を提供する。 Based on the above principle, the present invention provides a method for screening or identifying a substance that causes cross-adaptation of a target odor and a substance that suppresses the target odor by cross-adaptation.
 したがって本発明は、標的の匂いの交差順応を引き起こす物質の選択方法であって、
(1)嗅覚受容体ポリペプチドを探索し、標的の匂いの原因物質に応答するものを同定すること、
(2)該同定された嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該嗅覚受容体ポリペプチドの応答を活性化させた該試験物質を、該標的の匂いの交差順応を引き起こす物質として選択すること、
を含む方法を提供する。
Accordingly, the present invention is a method for selecting a substance that causes cross-adaptation of a target odor,
(1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent;
(2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
A method comprising:
 また本発明は、標的の匂いを抑制する物質の選択方法であって、
(1)嗅覚受容体ポリペプチドを探索し、標的の匂いの原因物質に応答するものを同定すること、
(2)該同定された嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該嗅覚受容体ポリペプチドの応答を活性化させた該試験物質を、該標的の匂いを抑制する物質として選択すること、
を含む方法を提供する。
The present invention also provides a method for selecting a substance that suppresses the target odor,
(1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent;
(2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that suppresses the odor of the target;
A method comprising:
 また本発明は、標的の匂いの交差順応を引き起こす物質の選択方法であって、
(1)標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドを準備すること、
(2)該少なくとも1種の嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該少なくとも1種の嗅覚受容体ポリペプチドのいずれかの応答を活性化させた該試験物質を、該標的の匂いの交差順応を引き起こす物質として選択すること、
を含む方法を提供する。
The present invention also provides a method for selecting a substance that causes cross-adaptation of a target odor,
(1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent;
(2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
A method comprising:
 また本発明は、標的の匂いを抑制する物質の選択方法であって、
(1)標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドを準備すること、
(2)該少なくとも1種の嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該少なくとも1種の嗅覚受容体ポリペプチドのいずれかの応答を活性化させた該試験物質を、該標的の匂いを抑制する物質として選択すること、
を含む方法を提供する。
The present invention also provides a method for selecting a substance that suppresses the target odor,
(1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent;
(2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that suppresses the target odor;
A method comprising:
 上記本発明の方法は、匂いの情報伝達を担う全ての嗅覚受容体ポリペプチド、及びそれらに認識される全ての匂いに適用することができる。 The above-described method of the present invention can be applied to all olfactory receptor polypeptides responsible for smell transmission and all odors recognized by them.
 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。上記本発明の方法においては、まず、交差順応を引き起こしたい又は抑制したい標的の匂いについて、該標的の匂いの原因物質に応答性を有する少なくとも1種の嗅覚受容体ポリペプチドを準備する。 The method of the present invention may be a method performed in vitro or ex vivo. In the above-described method of the present invention, first, for a target odor to cause or suppress cross-adaptation, at least one olfactory receptor polypeptide having responsiveness to a causative substance of the target odor is prepared.
 本発明で使用される、標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドは、嗅覚受容体ポリペプチドの集団を探索し、その中から、該標的の匂いの原因物質に応答するものを同定することによって得ることができる。同定すべき該「標的匂いの原因物質に対する嗅覚受容体」は、該標的匂いの原因物質の受容能力をもつ全ての嗅覚受容体である必要はなく、該標的匂いの認識に大きな役割をもつ受容体、言い換えれば該標的匂いの原因物質に対して比較的低い濃度から応答できる、もしくはある濃度の匂いの原因物質に対して比較的高い応答性を示す受容体であればよい。また、受容体を発現させた細胞応答の匂いの原因物質濃度依存性を求め、その結果より算出される50%効果濃度(EC50)が比較的低いものを「標的匂いの原因物質に対する嗅覚受容体」として同定してもよい。または、受容体ポリペプチドに対する匂いの原因物質の結合を直接的に評価する場合、同定すべき「標的匂いの原因物質に対する嗅覚受容体」は、解離定数が低いなどより高い結合能をもつ受容体であればよい。 As used in the present invention, at least one olfactory receptor polypeptide that responds to a target odor-causing agent is searched for a group of olfactory receptor polypeptides from which the odor-causing agent is targeted. It can be obtained by identifying what responds. The “olfactory receptor for the target odor causative agent” to be identified does not have to be all olfactory receptors capable of accepting the target odor causative agent, and is a receptor that plays a major role in the recognition of the target odor. Any receptor that can respond to the body, in other words, the causative substance of the target odor from a relatively low concentration, or exhibits a relatively high response to the causative substance of a certain concentration of odor may be used. Further, the dependence of the cellular response in which the receptor is expressed on the odor causative substance concentration is obtained, and the 50% effective concentration (EC50) calculated from the result is expressed as “an olfactory receptor for the causative substance of the target odor”. May be identified. Alternatively, when directly evaluating the binding of an odor causative agent to a receptor polypeptide, the “olfactory receptor for the target odor causative agent” to be identified is a receptor having a higher binding ability such as a low dissociation constant If it is.
 例えば、標的の匂いの原因物質に応答性を有する嗅覚受容体ポリペプチドは、後述の実施例1に記載されるように、参考例1~2記載の方法に従って種々の嗅覚受容体ポリペプチドによる応答をモニターし、標的の匂いの原因物質に応答を示す嗅覚受容体ポリペプチドをスクリーニングすることによって、同定することができる。 For example, an olfactory receptor polypeptide having responsiveness to a target odor causative substance is a response by various olfactory receptor polypeptides according to the method described in Reference Examples 1 and 2, as described in Example 1 described later. Can be identified by screening olfactory receptor polypeptides that respond to the causative agent of the target odor.
 上記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、哺乳動物由来の嗅覚受容体ポリペプチドであればよい。哺乳動物由来の嗅覚受容体ポリペプチドの好ましい例としては、ヒト、チンパンジーをはじめとする霊長類、又は、マウス、ラットなどげっ歯類由来の嗅覚受容体ポリペプチドが挙げられ、より好ましい例としては、ヒトがもつ400以上の嗅覚受容体及びそれと同等の機能を有するポリペプチドが挙げられる。ヒト、マウス及びラットの嗅覚受容体の情報は、GenBank[www.ncbi.nlm.nih.gov]より取得することができる。 The olfactory receptor polypeptide used in the method of the present invention may be any mammal-derived olfactory receptor polypeptide. Preferred examples of mammal-derived olfactory receptor polypeptides include primates such as humans, chimpanzees, or olfactory receptor polypeptides derived from rodents such as mice and rats. And 400 or more olfactory receptors possessed by humans and polypeptides having functions equivalent thereto. Information on human, mouse and rat olfactory receptors can be found in GenBank [www. ncbi. nlm. nih. gov].
 上記探索の対象となる嗅覚受容体ポリペプチドの集団は、単一の哺乳動物種由来であってもよく、又は2種以上の異なる哺乳動物種に由来する嗅覚受容体ポリペプチドを含んでいてもよい。好ましくは、上記探索の対象となる嗅覚受容体ポリペプチドの集団は、ヒト、マウス及びラットの嗅覚受容体、ならびにそれらとアミノ酸配列において少なくとも80%同一であってかつ嗅覚受容体と同等の機能を有するポリペプチドのうちのいずれかを含み、より好ましくは、ヒト嗅覚受容体、及びそれらとアミノ酸配列において少なくとも80%同一であってかつ嗅覚受容体と同等の機能を有するポリペプチドのうちのいずれかを含む。 The population of olfactory receptor polypeptides to be searched may be derived from a single mammalian species, or may contain olfactory receptor polypeptides derived from two or more different mammalian species. Good. Preferably, the population of olfactory receptor polypeptides to be searched is human, mouse and rat olfactory receptors, and at least 80% identical in amino acid sequence and having functions equivalent to those of olfactory receptors. More preferably, any of human olfactory receptors and polypeptides that are at least 80% identical in amino acid sequence to them and have functions equivalent to those of olfactory receptors including.
 本発明の方法において、上記嗅覚受容体ポリペプチドは、標的匂いの原因物質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、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、該嗅覚受容体ポリペプチドを天然に発現する組織若しくは細胞、又はそれらの培養物;該嗅覚受容体ポリペプチドを担持した嗅細胞の膜;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;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;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 target odor causing substance. 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 genetically engineered to express the olfactory receptor polypeptide or a culture thereof; 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、ヒト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。 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 human olfactory receptor polypeptides.
 上記組換え細胞は、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。あるいは細胞内に遺伝子の転写産物を直接導入することによっても嗅覚受容体ポリペプチドを発現させることが可能である。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、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、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タンパク質である。 The recombinant cell can be prepared by transforming a cell using a vector incorporating a gene encoding an olfactory receptor polypeptide. Alternatively, the olfactory receptor polypeptide can also be expressed by directly introducing a gene transcription product into a cell. 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 registered in GenBank as GI: 50234917.
 標的の匂いの種類は、特に限定されず、一般的に知られる悪臭又は不快臭(例えば、体臭、腋臭、口臭、糞便臭、尿臭、タバコ臭、カビ臭、生乾き臭、腐敗臭、生ごみ臭、汚水臭、排気臭、ダクト臭、排ガス臭、等)だけでなく、食品由来又は香料物質の匂い、その他の物質(例えば、化粧品、医薬品、洗浄剤、日用品、等)由来の匂い、などのあらゆる匂いを包含する。 The type of target odor is not particularly limited, and generally known bad odors or unpleasant odors (for example, body odor, habit odor, halitosis, fecal odor, urine odor, tobacco odor, mold odor, dry odor, rot odor, garbage Odors, sewage odors, exhaust odors, duct odors, exhaust gas odors, etc.) as well as odors from foods or fragrance materials, odors from other substances (eg cosmetics, pharmaceuticals, cleaning agents, daily necessities, etc.), etc. Includes all the odors.
 標的の匂いの原因物質は、嗅覚受容体に作用して該標的の匂いを知覚させる物質であればよい。該原因物質は、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。好ましくは、該原因物質は揮発性物質である。該原因物質の例としては、ムスク香の原因物質であるムスコン、タバコ臭の原因物質であるグアイアコール、尿臭の原因物質であるp-クレゾール、生乾き臭の原因物質である4-メチル-3-ヘキセン酸、腋臭の原因物質である3-メルカプト-3-メチルヘキサノール、3-ヒドロキシ-3-メチルヘキサン酸及び3-メチル-2-ヘキセン酸、カビ臭の原因物質であるジオスミン及び2-メチルイソボルネオール、糞便臭又は口臭の原因物質であるスカトール及びインドール、体臭の原因物質であるノナン酸、ヘキサン酸及びイソ吉草酸、生ごみ、汚水又は排水口から発せられる悪臭の原因物質である揮発性硫黄化合物、ならびにアクリル酸ブチル、ピラジン誘導体、フラネオール、ソトロンなどが挙げられる。 The causative substance of the target odor may be any substance that acts on the olfactory receptor to perceive the target odor. The causative substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, or a compound, a composition or a mixture. Also good. Preferably, the causative substance is a volatile substance. Examples of the causative agent include muscone, a causative agent of musk odor, guaiacol, a causative agent of tobacco odor, p-cresol, a causative agent of urine odor, 4-methyl-3- Hexenoic acid, 3-mercapto-3-methylhexanol, 3-hydroxy-3-methylhexanoic acid and 3-methyl-2-hexenoic acid, causative substances of odor, diosmine and 2-methyliso Borneol, skatole and indole that cause fecal odor or bad breath, nonanoic acid, hexanoic acid and isovaleric acid that cause body odor, volatile sulfur that is a cause of bad odor emitted from garbage, sewage or drainage Compounds, and butyl acrylate, pyrazine derivatives, furaneol, sotron and the like.
 上記嗅覚受容体ポリペプチドに標的の匂いの原因物質が添加され、該原因物質に対するそ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。したがって、該原因物質添加後の細胞内cAMP量を指標にすることで、該該原因物質に対する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、レポータージーンアッセイ等が挙げられる。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、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、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。 The causative substance of the target odor is added to the olfactory receptor polypeptide, and its response to the causative substance 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 an 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. Therefore, the response of the olfactory receptor polypeptide to the causative substance can be measured by using the amount of intracellular cAMP after the causative substance is added as an index. Examples of the method for measuring the amount of cAMP include an ELISA method and a reporter gene assay. Another method for measuring the response of an olfactory receptor polypeptide includes a calcium imaging method. Yet another method includes measurement by electrophysiological techniques. In electrophysiological measurement, for example, a cell (such as Xenopus oocyte) in which an 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 measurement using an electrode membrane potential fixation method or the like.
 次いで測定された応答に基づいて、上記標的の匂いの原因物質に応答する嗅覚受容体ポリペプチドを同定する。応答性の評価は、該原因物質を添加した該嗅覚受容体ポリペプチド(試験群)の応答を、対照群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、異なる濃度の該原因物質を添加した該嗅覚受容体ポリペプチド、該原因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、該原因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。あるいは、対照群としては、該原因物質に対して応答性を有さないもしくは低い応答性を有する他の嗅覚受容体ポリペプチドが挙げられる。 Then, based on the measured response, an olfactory receptor polypeptide that responds to the target odor-causing substance is identified. Evaluation of responsiveness can be performed by comparing the response of the olfactory receptor polypeptide (test group) to which the causative substance is added with the control group. The control group includes the olfactory receptor polypeptide to which the causative substance at different concentrations was added, the olfactory receptor polypeptide to which the causative substance was not added, the olfactory receptor polypeptide to which a control substance was added, and the cause The olfactory receptor polypeptide before adding a substance, cells in which the olfactory receptor polypeptide is not expressed, and the like can be mentioned. Alternatively, the control group includes other olfactory receptor polypeptides having no response or low response to the causative agent.
 試験群における応答が対照群よりも増強されていた場合、該嗅覚受容体ポリペプチドは、標的の匂いの原因物質に応答するものとして同定される。例えば、試験群における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200%以上、より好ましくは300%以上、さらに好ましくは400%以上に増強されていれば、該嗅覚受容体ポリペプチドは、標的の匂いの原因物質に応答するものとして同定される。あるいは、試験群における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増強されていれば、該嗅覚受容体ポリペプチドは、標的の匂いの原因物質に応答するものとして同定される。 If the response in the test group is enhanced over the control group, the olfactory receptor polypeptide is identified as responding to the target odor causative agent. For example, if the response of the olfactory receptor polypeptide in the test group is enhanced to 200% or more, more preferably 300% or more, and even more preferably 400% or more compared to the control group, the olfactory receptor is enhanced. Polypeptides are identified as responding to the target odor causative agent. Alternatively, if the response of the olfactory receptor polypeptide in the test group is statistically significantly enhanced as compared to the control group, the olfactory receptor polypeptide is responsive to the target odor causing substance. Identified as
 必要に応じて、同定された嗅覚受容体ポリペプチドに標的の匂いの原因物質を異なる濃度で添加して、同様の手順で応答を測定してもよい。応答が原因物質の濃度依存的に増加していれば、該嗅覚受容体ポリペプチドが標的の匂いの原因物質に応答性を有するものであると確認することができる。 If necessary, the target odor causative substance may be added to the identified olfactory receptor polypeptide at a different concentration, and the response may be measured by the same procedure. If the response increases in a concentration-dependent manner of the causative agent, it can be confirmed that the olfactory receptor polypeptide is responsive to the causative agent of the target odor.
 標的の匂いの原因物質に応答性を有する嗅覚受容体ポリペプチドが複数種見出され、それらの応答強度が互いに異なる場合、その中から、該原因物質に対して比較的高い応答性を示す1種以上の嗅覚受容体ポリペプチドをさらに選択することができる。例えば、標的の匂いの原因物質に対する応答性の高い方から1種、2種もしくは3種以上の嗅覚受容体ポリペプチドを選択することができ、またはさらに応答性の最も高い1種を選択することができる。あるいは、標的の匂いの原因物質に最も低い応答性を有する嗅覚受容体ポリペプチドを選択しないことができ、またはさらに応答性の低い方から2種以上を選択しないことができる。好適には、上記「選択する」1種以上の嗅覚受容体ポリペプチドは、上記「選択しない」嗅覚受容体ポリペプチドの各々と比べて、200%以上、好ましくは300%以上、より好ましくは400%以上の応答性を有する。あるいは、嗅覚受容体ポリペプチドの標的の匂いの原因物質に対する応答感度をEC50、応答閾値などを指標として評価し、高感度の嗅覚受容体ポリペプチドを選択することができる。さらに、上記応答性および応答感度の評価に基づいて、高感度かつ高応答性の嗅覚受容体ポリペプチドを選択することができる。 When multiple types of olfactory receptor polypeptides having responsiveness to a target odor causative agent are found and their response intensities are different from each other, a relatively high responsiveness to the causative agent is shown among them 1 More than one species of olfactory receptor polypeptide can be further selected. For example, one, two, or three or more olfactory receptor polypeptides can be selected from the ones with the highest responsiveness to the target odor-causing substance, or the one with the highest responsiveness is selected. Can do. Alternatively, the olfactory receptor polypeptide having the lowest responsiveness to the target odor-causing substance can not be selected, or two or more of the responsiveness can be not selected. Suitably, the “selected” one or more olfactory receptor polypeptides are 200% or more, preferably 300% or more, more preferably 400, compared to each of the “non-selected” olfactory receptor polypeptides. % Responsiveness. Alternatively, the sensitivity of the olfactory receptor polypeptide to the target odor causative substance can be evaluated using EC50, response threshold, etc. as an index, and a highly sensitive olfactory receptor polypeptide can be selected. Furthermore, a highly sensitive and highly responsive olfactory receptor polypeptide can be selected based on the evaluation of responsiveness and response sensitivity.
 嗅覚受容体ポリペプチドは、培養細胞などに発現させた際、その種類によって異なる基底活性を示す。そのため、嗅覚受容体ポリペプチド間で応答を比較する場合、嗅覚受容体ポリペプチドごとに応答値を基準化し、得られた基準化値について嗅覚受容体ポリペプチド間での比較を行うことが望ましい。基準化の方法としては、例えば、1種類の嗅覚受容体を発現させた細胞について、匂い刺激を行わなかった際のシグナル値を1としたときの匂い刺激に対するシグナルの相対値を求める方法、1種類の嗅覚受容体を発現させた細胞について、匂い刺激を行わなかった際のシグナル値を、匂い刺激に対するシグナル値から減算する方法、などが挙げられる。 Olfactory receptor polypeptides exhibit different basal activities depending on the type when expressed in cultured cells. Therefore, when comparing responses between olfactory receptor polypeptides, it is desirable to standardize the response values for each olfactory receptor polypeptide and compare the obtained normalized values between olfactory receptor polypeptides. As a standardization method, for example, for a cell expressing one type of olfactory receptor, a relative value of a signal with respect to an odor stimulation when the signal value when no odor stimulation is performed is set to 1, 1 For example, a method of subtracting a signal value when no odor stimulation is performed from a signal value with respect to the odor stimulation for cells expressing various types of olfactory receptors.
 以上の手順で、標的の匂いの原因物質に応答性を有する嗅覚受容体ポリペプチドを同定することができる。斯くして、標的の匂いの原因物質に応答性を有する少なくとも1種の嗅覚受容体ポリペプチドが準備される。 Through the above procedure, an olfactory receptor polypeptide having responsiveness to the target odor-causing substance can be identified. Thus, at least one olfactory receptor polypeptide that is responsive to the target odor-causing substance is prepared.
 本発明の方法において準備される、上記少なくとも1種の嗅覚受容体ポリペプチドの例としては、標的の匂いの原因物質に応答性を有するヒト、マウス又はラットの嗅覚受容体、及びそれとアミノ酸配列において少なくとも80%同一であって、かつ該標的の匂いの原因物質に対して応答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種が挙げられる。より好ましい例としては、標的の匂いの原因物質に応答性を有するヒト嗅覚受容体、及びそれとアミノ酸配列において少なくとも80%同一であって、かつ該標的の匂いの原因物質に対して応答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種が挙げられる。さらに好ましい例としては、標的の匂いの原因物質に応答性を有するヒト嗅覚受容体群のうち、最も高い応答性を示す嗅覚受容体、及びそれとアミノ酸配列において少なくとも80%同一であって、かつ該標的の匂いの原因物質に対して応答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種が挙げられる。なお好ましい例としては、標的の匂いの原因物質に応答性を有するヒト嗅覚受容体群のうち、最も高感度かつ高応答性を示す嗅覚受容体、及びそれとアミノ酸配列において少なくとも80%同一であって、かつ該標的の匂いの原因物質に対して応答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種が挙げられる。 Examples of the at least one olfactory receptor polypeptide prepared in the method of the present invention include a human, mouse or rat olfactory receptor responsive to a causative agent of a target odor, and an amino acid sequence thereof. And at least one selected from the group consisting of polypeptides that are at least 80% identical and responsive to the target odor causative agent. As a more preferred example, a human olfactory receptor that is responsive to a target odor causative agent, and at least 80% identical in amino acid sequence thereto, and responsive to the target odor causative agent Examples include at least one selected from the group consisting of polypeptides. Further preferred examples include a group of human olfactory receptors that are responsive to a target odor-causing substance, the olfactory receptors having the highest responsiveness, and at least 80% identical in amino acid sequence, and Examples thereof include at least one selected from the group consisting of polypeptides having responsiveness to a target odor causing substance. As a preferable example, among the human olfactory receptor group having responsiveness to the target odor-causing substance, the most sensitive and highly responsive olfactory receptor and the amino acid sequence thereof are at least 80% identical. And at least one selected from the group consisting of polypeptides that are responsive to the causative agent of the target odor.
 本発明の方法において準備される上記嗅覚受容体ポリペプチドは、上記標的匂いの原因物質に対する応答性を示す少なくとも1種であればよいが、いずれか2種以上を組み合わせてもよい。 The olfactory receptor polypeptide prepared in the method of the present invention may be at least one responsive to the target odor causative substance, or any two or more of them may be combined.
 例えば、本発明の方法において、標的匂いがムスク香である場合、匂い原因物質はムスク系香料であり、使用される嗅覚受容体ポリペプチドは、OR5AN1(配列番号2)、及び配列番号2で示されるアミノ酸配列と少なくとも80%同一なアミノ酸配列からなりかつムスク系香料に対して応答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(図1参照)。 For example, in the method of the present invention, when the target odor is a musk fragrance, the odor-causing substance is a musk fragrance, and the olfactory receptor polypeptide used is represented by OR5AN1 (SEQ ID NO: 2) and SEQ ID NO: 2. And at least one selected from the group consisting of polypeptides having a responsiveness to musk fragrances (see FIG. 1).
 また例えば、本発明の方法において、標的匂いがグアイアコール(2-メトキシフェノール)の匂いである場合、匂い原因物質はグアイアコールであり、使用される嗅覚受容体ポリペプチドは、OR10G4(配列番号4)、及び配列番号4で示されるアミノ酸配列と少なくとも80%同一なアミノ酸配列からなりかつグアイアコールに対して応答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(図1参照)。 Also, for example, in the method of the present invention, when the target odor is guaiacol (2-methoxyphenol), the odor-causing substance is guaiacol, and the olfactory receptor polypeptide used is OR10G4 (SEQ ID NO: 4), And at least one selected from the group consisting of polypeptides comprising an amino acid sequence at least 80% identical to the amino acid sequence shown in SEQ ID NO: 4 and having responsiveness to guaiacol (see FIG. 1).
 グアイアコールは、タバコ臭の主な原因物質であることが知られている(特開2006-321943号公報)。したがって、本発明の方法の一実施形態においては、標的匂いはタバコ臭であり、使用される嗅覚受容体ポリペプチドは、グアイアコールの受容体であるOR10G4(配列番号4)、及び配列番号4で示されるアミノ酸配列と少なくとも80%同一なアミノ酸配列からなりかつタバコ臭原因物質、好ましくはグアイアコールに対して応答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。 Guaiacol is known to be a main causative substance of tobacco odor (Japanese Patent Laid-Open No. 2006-321944). Therefore, in one embodiment of the method of the present invention, the target odor is tobacco odor, and the olfactory receptor polypeptide used is represented by OR10G4 (SEQ ID NO: 4), which is a receptor for guaiacol, and SEQ ID NO: 4. And at least one selected from the group consisting of polypeptides that are responsive to tobacco odor-causing substances, preferably guaiacol.
 本発明の方法の一実施形態において、標的匂いは尿臭である。特開2009-132770号公報には、飛び散って乾燥した尿、又は使用後放置したおむつから発する尿臭に対する最も寄与の高い成分がp-クレゾールであること、及びp-クレゾールとそれ以外の炭素数6~10のフェノール化合物との組成物により、尿臭を再現できることが記載されている。国際公開公報第2009/037861号には、菌が産生するβ-グルクロニダーゼが尿に作用すると、p-クレゾール又はその他の成分が尿中に増加し、尿臭強度を顕著に増加させることが記載されている。さらに本出願人は、p-クレゾールに応答する嗅覚受容体の応答性を抑える物質により、尿臭を抑制できることを見出し、特許出願を行っている(特願2015-060636)。したがって、本発明における「尿臭原因物質」の例としては、尿のβ-グルクロニダーゼ処理物又はその抽出物、及びp-クレゾールが挙げられる。尿のβ-グルクロニダーゼ処理物としては、尿にβ-グルクロニダーゼを添加して処理したもの、尿中の菌が産生するβ-グルクロニダーゼが作用した尿、などが挙げられる。これらの尿処理物又はその抽出物は、p-クレゾールを含有する。 In one embodiment of the method of the present invention, the target odor is urine odor. JP-A-2009-132770 discloses that p-cresol is the component that has the highest contribution to urine odor emitted from scattered and dried urine or diapers left after use, and p-cresol and other carbon numbers. It is described that a urine odor can be reproduced by a composition with 6 to 10 phenolic compounds. International Publication No. 2009/037861 describes that when β-glucuronidase produced by bacteria acts on urine, p-cresol or other components increase in the urine, and the urine odor intensity is significantly increased. ing. Furthermore, the present applicant has found that a urine odor can be suppressed by a substance that suppresses olfactory receptor responsiveness to p-cresol, and has filed a patent application (Japanese Patent Application No. 2015-060636). Therefore, examples of the “urine odor-causing substance” in the present invention include urinary β-glucuronidase-treated product or an extract thereof, and p-cresol. Examples of the urinary β-glucuronidase-treated product include those obtained by adding β-glucuronidase to urine and urine on which β-glucuronidase produced by bacteria in the urine acts. These urine processed products or extracts thereof contain p-cresol.
 尿臭原因物質であるp-クレゾールを認識する嗅覚受容体としては、OR9Q2が見出されている(図1及び2参照)。OR9Q2は、GenBankにGI:284413710として登録されている、配列番号6で示されるアミノ酸配列からなるポリペプチドである。したがって好ましくは、本発明の方法において標的匂いが尿臭である場合、使用される嗅覚受容体ポリペプチドは、OR9Q2(配列番号6)、及び配列番号6で示されるアミノ酸配列と少なくとも80%同一なアミノ酸配列からなりかつ尿臭原因物質、好ましくはp-クレゾールに対して応答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。 OR9Q2 has been found as an olfactory receptor that recognizes p-cresol, which is a urinary odor causative substance (see FIGS. 1 and 2). OR9Q2 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, which is registered as GI: 284413710 with GenBank. Therefore, preferably, when the target odor is urine odor in the method of the present invention, the olfactory receptor polypeptide used is at least 80% identical to OR9Q2 (SEQ ID NO: 6) and the amino acid sequence represented by SEQ ID NO: 6. It is at least one selected from the group consisting of an amino acid sequence and a polypeptide having responsiveness to a substance that causes urine odor, preferably p-cresol.
 次いで、本発明の方法においては、上記標的の匂いの原因物質に応答性を有する嗅覚受容体ポリペプチドに対して、試験物質を添加する。 Next, in the method of the present invention, a test substance is added to the olfactory receptor polypeptide having responsiveness to the target odor-causing substance.
 本発明の方法に使用される試験物質は、標的の匂いの交差順応を引き起こす物質として、又は標的の匂いの抑制物質として使用することを所望する物質であれば、特に限定されない。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。ただし試験物質は、標的の匂いの原因物質とは異なる物質である。好ましくは、試験物質は揮発性物質であって、標的匂いとは別の匂いを有する物質である。また好ましくは、試験物質は、標的匂いとは別の匂いを有する香料である。 The test substance used in the method of the present invention is not particularly limited as long as it is a substance that is desired to be used as a substance that causes cross-adaptation of the target odor or as a substance that suppresses the target odor. 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. However, the test substance is different from the causative substance of the target odor. Preferably, the test substance is a volatile substance and has a smell different from the target odor. Also preferably, the test substance is a fragrance having an odor different from the target odor.
 続いて、上記試験物質に対する嗅覚受容体ポリペプチドの応答が測定される。測定は、標的の匂いの原因物質に対する嗅覚受容体ポリペプチドの応答の測定に関して上記で説明した方法に従って行えばよい。 Subsequently, the response of the olfactory receptor polypeptide to the test substance is measured. The measurement may be performed according to the method described above with respect to the measurement of the response of the olfactory receptor polypeptide to the causative agent of the target odor.
 次いで、測定された嗅覚受容体ポリペプチドの応答に基づいて、上記試験物質の受容体活性化作用を評価し、標的の匂いの交差順応を引き起こす試験物質を同定する。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(試験群)の応答を、対照群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。あるいは、対照群としては、該試験物質に対して応答性を有さないもしくは低い応答性を有する他の嗅覚受容体ポリペプチドが挙げられる。 Next, based on the measured response of the olfactory receptor polypeptide, the receptor activation effect of the test substance is evaluated, and the test substance causing cross adaptation of the target odor is identified. The effect of the test substance can be evaluated by comparing the response of the olfactory receptor (test group) to which the test substance is added with 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. Alternatively, the control group includes other olfactory receptor polypeptides that have no response or low response to the test substance.
 試験群における応答が、対照群よりも増強されていた場合、当該試験物質を、該嗅覚受容体ポリペプチドの応答を活性化する物質として評価することができる。例えば、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、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、試験物質添加前後、又は嗅覚受容体ポリペプチド発現細胞群と非発現細胞群との間で、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。嗅覚受容体ポリペプチドの応答が、試験物質添加により誘導された場合、該試験物質は、標的匂いの原因物質に応答する嗅覚受容体ポリペプチドの応答を活性化する物質として評価される。 When the response in the test group is enhanced as compared with the control group, the test substance can be evaluated as a substance that activates the response of the olfactory receptor polypeptide. For example, the effect of the test substance on the response of the olfactory receptor polypeptide is between the test substance addition group and the non-addition group, between the test substance addition group and the control substance addition group, before and after the addition of the test substance, or It can be evaluated by comparing the response of the olfactory receptor polypeptide between the olfactory receptor polypeptide-expressing cell group and the non-expressing cell group. When the response of the olfactory receptor polypeptide is induced by addition of a test substance, the test substance is evaluated as a substance that activates the response of the olfactory receptor polypeptide in response to the causative substance of the target odor.
 例えば、試験物質添加群における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200%以上、より好ましくは300%以上、さらに好ましくは400%以上に増強されていれば、該試験物質を、嗅覚受容体ポリペプチドの応答を活性化する物質として評価することができる。あるいは、試験物質添加群における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増強されていれば、該試験物質を、嗅覚受容体ポリペプチドの応答を活性化する物質として評価することができる。 For example, if the response of the olfactory receptor polypeptide in the test substance addition group is enhanced to 200% or more, more preferably 300% or more, and further preferably 400% or more, compared to the control group, the test is performed. A substance can be evaluated as a substance that activates the response of an olfactory receptor polypeptide. Alternatively, if the response of the olfactory receptor polypeptide in the test substance-added group is statistically significantly enhanced as compared with the control group, the test substance activates the response of the olfactory receptor polypeptide. It can be evaluated as a substance.
 上記で得られた標的の匂いの原因物質に応答する嗅覚受容体ポリペプチドの応答を活性化する試験物質は、該標的の匂いの交差順応を引き起こす物質として選択される。すなわち、該試験物質の存在下では、標的匂いを認識する嗅覚受容体は、活性され、続いて順応により応答性が低下するので、後から添加される標的匂いの原因物質に対する応答性を損なう。結果、該試験物質によって標的匂いの交差順応が引き起こされる。 The test substance that activates the response of the olfactory receptor polypeptide in response to the target odor-causing substance obtained above is selected as a substance that causes cross-adaptation of the target odor. That is, in the presence of the test substance, the olfactory receptor that recognizes the target odor is activated and subsequently responsiveness decreases due to adaptation, so that the responsiveness to the target odor causing substance added later is impaired. As a result, cross-adaptation of the target odor is caused by the test substance.
 あるいは、上記で得られた標的の匂いの原因物質に応答する嗅覚受容体ポリペプチドの応答を活性化する試験物質は、該標的の匂いを抑制する物質として選択される。すなわち、該試験物質は、標的の匂いの交差順応を引き起こすことによって、該標的の匂いを抑制することができる物質である。 Alternatively, the test substance that activates the response of the olfactory receptor polypeptide that responds to the target odor causative substance obtained above is selected as a substance that suppresses the target odor. That is, the test substance is a substance that can suppress the target odor by causing cross adaptation of the target odor.
 本発明の方法においては、必要に応じて、上記手順にて選択された試験物質を官能試験にかけ、その交差順応能、又は標的の匂いの抑制能をさらに評価してもよい。官能試験は、当該分野で通常行われる消臭剤の評価手順に準じて行われ得るが、好ましくは、試験物質が交差順応誘導物質であることを考慮して、評価者に対する試験物質と標的の匂い原因物質の適用順序が調整される。例えば、本発明による官能試験では、評価者は、最初に上記手順にて選択された候補試験物質の匂いを嗅ぎ、その匂いに順応しておく。次いで該評価者は、標的の匂いを嗅ぎ、その強度を評価する。得られた評価結果は、試験物質に順応させなかった場合の標的の匂いの強度と比較される。官能試験の結果、標的の匂いの強度を低下させたと評価された試験物質は、該標的の匂いに対して交差順応を起こさせる物質、又は該標的の匂いを抑制する物質として選択される。 In the method of the present invention, if necessary, the test substance selected in the above procedure may be subjected to a sensory test to further evaluate its cross-adaptation ability or target odor suppression ability. The sensory test can be performed in accordance with a deodorant evaluation procedure normally performed in the field, but preferably, the test substance and target for the evaluator are considered in consideration of the test substance being a cross-adaptation inducer. The order of application of odor-causing substances is adjusted. For example, in the sensory test according to the present invention, the evaluator first smells the candidate test substance selected in the above procedure and adapts to the smell. The evaluator then smells the target and evaluates its intensity. The evaluation results obtained are compared with the intensity of the target odor when not adapted to the test substance. As a result of the sensory test, the test substance evaluated as having reduced the intensity of the target odor is selected as a substance that causes cross adaptation to the target odor or a substance that suppresses the target odor.
 本発明の方法によって選択された物質は、交差順応に基づいて標的の匂いを抑制することができる物質である。本発明の方法によって得られた標的の匂いを抑制する物質を使用する場合の一実施形態は、以下のとおりである:まず、標的の匂いの抑制を所望する対象者に、該対象者が標的の匂いに曝露される前に、該標的の匂いを抑制する物質の匂いを嗅がせておく。あるいは、該対象者に対し、標的の匂いよりも強い匂いとなるように該標的の匂いを抑制する物質を適用する。その結果、該対象者は、標的の匂いに曝露されても、該標的の匂いに対する嗅覚感受性が低下しているため、該標的の匂いを弱いと感じるか、又は感じなくなる。本発明で得られた標的の匂いを抑制する物質の適用例としては、トイレの前又は中への該物質の載置;病棟又は介護施設などで排泄の処置に関わる者に、該物質を携行させたり、該処置の前に該物質を曝露したりする方法;該物質を含んだ紙おむつ又は生理用品;該物質を含んだ肌着、下着、リネン類等の服飾類、布製品、又は織物;該物質を含んだ洗濯用洗剤又は柔軟剤;該物質を含んだ香粧品、洗浄剤、デオドラント等の外用剤、医薬品、食品、等;標的の匂いを有する製品の製造ライン若しくは標的の匂いを発生する環境への適用、などが挙げられるが、これらに限定されるものではない。 The substance selected by the method of the present invention is a substance that can suppress the target odor based on cross adaptation. One embodiment of using a substance that suppresses the target odor obtained by the method of the present invention is as follows: First, the target is targeted to a subject who desires suppression of the target odor. Before being exposed to the odor of the target, the odor of the substance that suppresses the target odor is smelled. Alternatively, a substance that suppresses the odor of the target is applied to the subject so that the odor is stronger than the odor of the target. As a result, even if the subject is exposed to the target odor, the odor sensitivity to the target odor is reduced, and therefore the target odor is felt weak or no longer felt. Examples of the application of the substance that suppresses the target odor obtained in the present invention include placing the substance in front of or in a toilet; carrying the substance to a person involved in excretion treatment in a ward or a nursing facility Or a method of exposing the substance before the treatment; a paper diaper or a sanitary product containing the substance; an undergarment, underwear, linen or other clothing containing the substance, a cloth product, or a fabric; Laundry detergents or softeners containing substances; cosmetics, detergents, deodorants and other external preparations containing the substances, pharmaceuticals, foods, etc .; production lines for products with a target odor or target odors generated Examples include, but are not limited to, application to the environment.
 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>標的の匂いの交差順応を引き起こす物質の選択方法であって、
(1)嗅覚受容体ポリペプチドを探索し、標的の匂いの原因物質に応答するものを同定すること、
(2)該同定された嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該嗅覚受容体ポリペプチドの応答を活性化させた該試験物質を、該標的の匂いの交差順応を引き起こす物質として選択すること、
を含む、方法。
<1> A method for selecting a substance that causes cross-adaptation of a target odor,
(1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent;
(2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
Including the method.
<2>標的の匂いを抑制する物質の選択方法であって、
(1)嗅覚受容体ポリペプチドを探索し、標的の匂いの原因物質に応答するものを同定すること、
(2)該同定された嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該嗅覚受容体ポリペプチドの応答を活性化させた該試験物質を、該標的の匂いを抑制する物質として選択すること、
を含む、方法。
<2> A method for selecting a substance that suppresses a target odor,
(1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent;
(2) adding a test substance to the identified olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that suppresses the odor of the target;
Including the method.
<3>好ましくは、上記(1)で探索される嗅覚受容体ポリペプチドが、ヒト、マウス及びラットの嗅覚受容体、ならびにそれらとアミノ酸配列において少なくとも80%同一であってかつ嗅覚受容体と同等の機能を有するポリペプチドのうちのいずれかを含む、嗅覚受容体ポリペプチドの集団である、<1>又は<2>記載の方法。 <3> Preferably, the olfactory receptor polypeptide searched in (1) above is at least 80% identical in amino acid sequence to human, mouse and rat olfactory receptors and equivalent to the olfactory receptors <1> or <2> The method according to <1> or <2>, which is a population of olfactory receptor polypeptides comprising any of the polypeptides having the functions of:
<4>標的の匂いの交差順応を引き起こす物質の選択方法であって、
(1)標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドを準備すること、
(2)該少なくとも1種の嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該少なくとも1種の嗅覚受容体ポリペプチドのいずれかの応答を活性化させた該試験物質を、該標的の匂いの交差順応を引き起こす物質として選択すること、
を含む、方法。
<4> A method for selecting a substance that causes cross-adaptation of a target odor,
(1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent;
(2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
Including the method.
<5>標的の匂いを抑制する物質の選択方法であって、
(1)標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドを準備すること、
(2)該少なくとも1種の嗅覚受容体ポリペプチドに試験物質を添加して、その応答を測定すること、
(3)該少なくとも1種の嗅覚受容体ポリペプチドのいずれかの応答を活性化させた該試験物質を、該標的の匂いを抑制する物質として選択すること、
を含む、方法。
<5> A method for selecting a substance that suppresses a target odor,
(1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent;
(2) adding a test substance to the at least one olfactory receptor polypeptide and measuring the response;
(3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that suppresses the target odor;
Including the method.
<6>好ましくは、上記少なくとも1種の嗅覚受容体ポリペプチドが、以下からなる群より選択される少なくとも1種である、<4>又は<5>記載の方法:
 ラット若しくはマウス嗅覚受容体;
 ヒト嗅覚受容体;及び
 該ラット、マウス若しくはヒト嗅覚受容体とアミノ酸配列において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%以上同一であって、かつ上記標的の匂いの原因物質に対して応答性を有するポリペプチド。
<6> The method according to <4> or <5>, wherein the at least one olfactory receptor polypeptide is preferably at least one selected from the group consisting of:
Rat or mouse olfactory receptor;
Human olfactory receptor; and 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more in amino acid sequence with the rat, mouse or human olfactory receptor More preferably, the polypeptide is 99% or more identical and responsive to the target odor-causing substance.
<7>好ましくは、上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、<1>~<6>のいずれか1項記載の方法。 <7> Preferably, the olfactory receptor polypeptide is expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide, any of <1> to <6> The method according to claim 1.
<8>好ましくは、上記嗅覚受容体ポリペプチドの応答が、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、あるいはカルシウムイメージング又は電気生理学的手法により測定される、<1>~<7>のいずれか1項記載の方法。 <8> Preferably, the response of the olfactory receptor polypeptide is measured by intracellular cAMP amount measurement by ELISA or reporter gene assay, or by calcium imaging or electrophysiological technique, any one of <1> to <7> The method according to claim 1.
<9>好ましくは、(4)官能試験により、上記(3)で選択された物質の中から、上記標的の匂いに対して交差順応を起こさせる物質、又は上記標的の匂いを抑制する物質を選択すること、
をさらに含む、<1>~<8>のいずれか1項記載の方法。
<9> Preferably, (4) a substance that causes cross adaptation to the target odor or a substance that suppresses the target odor among the substances selected in (3) above by a sensory test. To choose,
The method according to any one of <1> to <8>, further comprising:
<10><1>~<9>のいずれか1項記載の方法であって、好ましくは、上記標的の匂いがタバコ臭又は尿臭である、方法。 <10> The method according to any one of <1> to <9>, wherein the target odor is preferably a tobacco odor or a urine odor.
<11><4>~<9>のいずれか1項記載の方法であって、好ましくは、
 上記標的の匂いがタバコ臭であり、かつ
 上記少なくとも1種の嗅覚受容体ポリペプチドが、以下:
  配列番号4で示されるアミノ酸配列からなるポリペプチド;及び
  配列番号4で示されるアミノ酸配列と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%以上同一であって、かつタバコ臭原因物質に対して応答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、方法。
<11> The method according to any one of <4> to <9>, preferably,
The target odor is tobacco odor, and the at least one olfactory receptor polypeptide is:
A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4; and 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably, with the amino acid sequence represented by SEQ ID NO: 4 Is a polypeptide that is 98% or more, preferably 99% or more identical and responsive to tobacco odor-causing substances,
A method which is at least one selected from the group consisting of:
<12><4>~<9>のいずれか1項記載の方法であって、好ましくは、
 上記標的の匂いが尿臭であり、かつ
 上記少なくとも1種の嗅覚受容体ポリペプチドが、以下:
  配列番号6で示されるアミノ酸配列からなるポリペプチド;及び
  配列番号6で示されるアミノ酸配列と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%以上同一であって、かつ尿臭原因物質に対して応答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、方法。
<12> The method according to any one of <4> to <9>, preferably,
The target odor is urine odor, and the at least one olfactory receptor polypeptide is:
A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6; and 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably, with the amino acid sequence represented by SEQ ID NO: 6 Is a polypeptide which is 98% or more, preferably 99% or more, and has a response to a urine odor causing substance,
A method which is at least one selected from the group consisting of:
<13><1>~<12>のいずれか1項記載の方法であって、好ましくは、上記試験物質が上記標的の匂いの原因物質とは異なる物質である、方法。 <13> The method according to any one of <1> to <12>, wherein the test substance is preferably a substance different from the target odor causing substance.
<14><1>~<13>のいずれか1項記載の方法であって、好ましくは、上記標的の匂いの原因物質に対して応答性を示す少なくとも1種の嗅覚受容体ポリペプチドの中から、該原因物質に対して高い応答性を示すものを選択し、標的の匂いの原因物質に応答する嗅覚受容体ポリペプチドとして同定することをさらに含む、方法。 <14> The method according to any one of <1> to <13>, preferably, among at least one olfactory receptor polypeptide exhibiting responsiveness to the target odor-causing substance. The odorant receptor polypeptide that responds to the causative agent of the target odor is further selected.
 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.
参考例1 ヒト嗅覚受容体発現細胞の調製
1)ヒト嗅覚受容体遺伝子のクローニング
 GenBankに登録されている配列情報を基に、表1-1及び1-2に記載の370種のヒト嗅覚受容体をコードする各遺伝子、及びヒト嗅覚受容体OR5AN1、OR10G4、OR9Q2をコードする遺伝子(それぞれ、配列番号1、3、5)をクローニングした。各遺伝子は、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。
Reference Example 1 Preparation of cells expressing human olfactory receptor 1) Cloning of human olfactory receptor gene 370 human olfactory receptors listed in Tables 1-1 and 1-2 based on sequence information registered in GenBank And genes encoding human olfactory receptors OR5AN1, OR10G4, and OR9Q2 (SEQ ID NOs: 1, 3, and 5 respectively) were cloned. Each gene was cloned by PCR using human genomic DNA female (G1521: Promega) as 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.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
2)pME18S-ヒトRTP1Sベクターの作製
 ヒトRTP1Sをコードする遺伝子をpME18SベクターのEcoRI、XhoIサイトへ組み込んだ。
2) Preparation of pME18S-human RTP1S vector A gene encoding human RTP1S 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
 実施例1の1)では、ヒト嗅覚受容体370種をそれぞれ発現させた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、384ウェルプレート(BioCoat)の各ウェルに4.4μLずつ添加した。次いで、HEK293細胞(20×104細胞/cm2)を40μL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。
3) Production of olfactory receptor-expressing cells In Example 1 1), HEK293 cells each expressing 370 types of human olfactory receptors were produced. 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 4.4 μL was added to each well of a 384 well plate (BioCoat). Next, 40 μL of HEK293 cells (20 × 10 4 cells / cm 2 ) were seeded in each well and cultured for 24 hours in an incubator maintained at 37 ° C. and 5% CO 2 .
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
 実施例1の2)及び実施例2、3では、ヒト嗅覚受容体OR5AN1、OR10G4、OR9Q2又はOR1A1を発現させたHEK293細胞を作製した。表3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(BioCoat)の各ウェルに10μLずつ添加した。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。対照区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。 In Example 1 2) and Examples 2 and 3, HEK293 cells expressing human olfactory receptors OR5AN1, OR10G4, OR9Q2 or OR1A1 were prepared. A reaction solution having the composition shown in Table 3 was prepared and allowed to stand in a clean bench for 15 minutes, and then 10 μL was added to each well of a 96-well plate (BioCoat). 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 . In order to use as a control group, cells (Mock) under conditions that do not express olfactory receptors were also prepared and used in the experiment in the same manner.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
参考例2 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、遺伝子導入効率又は細胞数の誤差を補正する内部標準として用いた。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。匂い物質刺激により誘導されたfLuc/hRlucを、匂い物質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。用量作用曲線の解析はGraphPad Prismを用いて行った。
Reference Example 2 Luciferase Assay The olfactory receptor expressed in HEK293 cells is conjugated 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). Further, a renilla luciferase gene fused downstream of the CMV promoter (hRluc-CMV) was simultaneously introduced, and used as an internal standard for correcting errors in gene transfer efficiency or cell number. 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 induced by odor substance stimulation by fLuc / hRluc in a cell not subjected to odor substance stimulation was calculated as a fold increase and used as an index of response intensity. Dose response curves were analyzed using GraphPad Prism.
参考例3 匂い物質
 匂い物質としては、以下を使用した。
 Muscone((R)-3- Methyl-1-cyclopentadecanone;MP BioMedical社)、
 Musk xylol(1-tert-Butyl-3,5-dimethyl-2,4,6-trinitrobenzene;東京化成工業社)、
 Muscenone delta(3-Methyl-5-cyclopentadecen-1-one、;FIRMENICH社)、
 Globanone(Cyclohexadec-8-en-1-one;SYMRISE社)、
 Exaltolide(登録商標)(16-Oxacyclohexadecan-1-one;FIRMENICH社)、
 Ambrettolide(17-Oxacycloheptadec-6-en-1-one;Sigma-Aldrich社)、
 Phenyl ethyl alcohol(PEA)(2-Phenylethanol;Sigma-Aldrich社)、
 Lyral(登録商標)(4-(4-Hydroxy-4-methyl-pentyl)-cyclohex-3-enecarbaldehyde;高砂香料社又はIFF社)
 Cis-3-hexenol(Sigma-Aldrich社)、
 Guaiacol(2-Methoxyphenol;東京化成工業社)、
 Ethylvanillin(3-Ethoxy-4-hydroxybenzaldehyde;Sigma-Aldrich社)、
 p-Cresol
 p-Cresyl acetate((4-Methylphenyl)acetate;東京化成工業社)、
 p-Cresyl iso butyrate(p-Tolyl isobutyrate、又は(4-Methylphenyl)2-methylpropanoate;東京化成工業社)、
 p-Methyl quinoline(6-Methyl quinoline;Sigma-Aldrich社)
Reference Example 3 Odor substance As the odor substance, the following was used.
Muscone ((R) -3-Methyl-1-cyclodecanedone; MP BioMedical),
Musk xylol (1-tert-Butyl-3,5-dimethyl-2,4,6-trinitrobenzene; Tokyo Chemical Industry Co., Ltd.),
Muscenene delta (3-Methyl-5-cyclopentadecen-1-one; FIRMENICH),
Globanone (Cyclohexadec-8-en-1-one; SYMRIS),
Exaltlide (registered trademark) (16-Oxacyclohexadecan-1-one; FIRMENIC),
Ambrettolide (17-Oxacycloheptadec-6-en-1-one; Sigma-Aldrich),
Phenyl ethyl alcohol (PEA) (2-Phenylethanol; Sigma-Aldrich),
Lyral (registered trademark) (4- (4-Hydroxy-4-methyl-pentyl) -cyclohex-3-enecarbaldehyde; Takasago International Corporation or IFF)
Cis-3-hexenol (Sigma-Aldrich),
Guiacol (2-Methoxyphenol; Tokyo Chemical Industry Co., Ltd.),
Ethylvanillin (3-Ethoxy-4-hydroxybenzaldehyde; Sigma-Aldrich),
p-Cresol
p-Cresyl acetate ((4-Methylphenyl) acetate; Tokyo Chemical Industry Co., Ltd.)
p-Cresyl iso butyrate (p-Tolyl isobutyrate, or (4-Methylphenyl) 2-methylpropanoate; Tokyo Chemical Industry Co., Ltd.),
p-Methyl quinoline (6-Methyl quinoline; Sigma-Aldrich)
実施例1 匂い物質に対する嗅覚受容体応答
1)匂い物質に応答する嗅覚受容体の同定
 参考例1に従って作製した嗅覚受容体発現細胞の培養物から培地を取り除き、匂い物質(Muscone、Guaiacol又はp-Cresol)を添加した。Muscone若しくはグアイアコールを含むDMEM培地(Nacalai)を、該培養物を含む384ウェルプレートの各ウェルに30μLずつ添加した(最終濃度:Muscone 100μM、Guaiacol 1mM)。またp-Cresolを含むCD293培地(Invitrogen)を、該培養物を含む96ウェルプレートの各ウェルに75μLずつ添加した(最終濃度1mM)。細胞をCO2インキュベータ内で2.5~3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後、参考例2の手法でルシフェラーゼアッセイを行い、匂い物質に対する嗅覚受容体の応答強度(fold increase)を測定した。
Example 1 Response of olfactory receptor to odorant 1) Identification of olfactory receptor responding to odorant The medium was removed from the culture of olfactory receptor-expressing cells prepared according to Reference Example 1, and the odorant (Muscone, Guiacol or p-) Cresol) was added. 30 μL of Muscone or Guaiacol-containing DMEM medium (Nacalai) was added to each well of the 384-well plate containing the culture (final concentrations: Muscone 100 μM, Guaicol 1 mM). In addition, 75 μL of CD293 medium (Invitrogen) containing p-Cresol was added to each well of a 96-well plate containing the culture (final concentration 1 mM). After culturing the cells in a CO 2 incubator for 2.5 to 3 hours and sufficiently expressing the luciferase gene in the cells, the luciferase assay is performed by the method of Reference Example 2 to determine the response intensity of the olfactory receptor to the odorant ( fold increase) was measured.
 結果を図1に示す。縦軸は、匂い刺激なしの条件での応答強度を1としたときの、各受容体発現細胞の匂い刺激に対する相対応答強度を表す。Muscone、Guaiacol及びp-Cresolに対して最も高い応答性をもたらす受容体として、OR5AN1、OR10G4、OR9Q2がそれぞれ同定された。 The results are shown in FIG. The vertical axis represents the relative response intensity of each receptor-expressing cell to the odor stimulation when the response intensity under the condition without odor stimulation is 1. OR5AN1, OR10G4, and OR9Q2 were identified as receptors that gave the highest responsiveness to Muscone, Guiacol, and p-Cresol, respectively.
2)嗅覚受容体応答の匂い物質濃度依存性
 参考例1、2記載の方法に従って、異なる濃度のp-Cresolに対するOR9Q2の応答を測定した。その結果、OR9Q2はp-クレゾール濃度依存的な応答を示し、p-Cresol受容体であることが確認された(図2)。
2) Odor substance concentration dependence of olfactory receptor response According to the methods described in Reference Examples 1 and 2, the response of OR9Q2 to different concentrations of p-Cresol was measured. As a result, OR9Q2 showed a p-cresol concentration-dependent response and was confirmed to be a p-Cresol receptor (FIG. 2).
実施例2 嗅覚受容体に対する匂い物質の作用
1)嗅覚受容体応答の測定
 参考例1に従って作製した嗅覚受容体発現細胞の培養物から、培地を取り除き、DMEM培地(ナカライテスク)で0.3μMから100μMに調製した匂い物質を含む溶液を75μL添加した。細胞をCO2インキュベータ内で3~4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後、参考例2の手法でルシフェラーゼアッセイを行い、匂い物質に対する嗅覚受容体の応答強度(fold increase)を測定した。
Example 2 Action of odorant substance on olfactory receptor 1) Measurement of olfactory receptor response The medium was removed from the culture of olfactory receptor-expressing cells prepared according to Reference Example 1, and from 0.3 μM in DMEM medium (Nacalai Tesque). 75 μL of a solution containing an odorant prepared to 100 μM was added. After culturing the cells in a CO 2 incubator for 3 to 4 hours to fully express the luciferase gene in the cells, the luciferase assay is performed by the method of Reference Example 2, and the response intensity of the olfactory receptor to the odorant (fold increase) ) Was measured.
2)結果
 OR5AN1は、ムスク系香料に応答する嗅覚受容体である。OR5AN1に対して9種の物質を適用した結果、その応答を活性化させる物質として、Musconeに加えて、Musk xylol、Muscenone delta、Globanone、及びExaltolide(登録商標)が見出された。OR5AN1は、これらの物質に対して濃度依存的に応答した(図3)。これらの応答は、OR5AN1を発現させない細胞(Mock)では認められなかったことから、OR5AN1に依存したものである。一方、Ambrettolide、Phenyl ethyl alcohol(PEA)、Lyral(登録商標)、及びCis-3-hexanolは、OR5AN1の応答を引き起こさなかった。
 一方で、Musconeに対してOR5AN1よりも弱い応答性を示したOR1A1(図1)は、PEA及びCis-3-hexanolによっても活性化された(図4)。
 OR10G4については、GuaiacolとEthylvanillinに対する応答性を有することが確認された(図5A)。
 OR9Q2については、図2にも示したとおり尿臭原因物質であるp-Cresolに対する応答性が確認されるとともに、さらにp-Cresyl Acetate、p-Cresyl iso butyrate、及びp-Methyl quinolineに対する応答性が見出された(図5B)。一方、OR10G4及びOR9Q2はいずれも、Musconeに対する応答性をもたないことが判明した(図5C)。
2) Results OR5AN1 is an olfactory receptor that responds to musk fragrances. As a result of applying nine kinds of substances to OR5AN1, in addition to Muscone, Musk xylol, Muscenene delta, Globanone, and Exaltolide (registered trademark) were found as substances that activate the response. OR5AN1 responded to these substances in a concentration-dependent manner (FIG. 3). These responses were dependent on OR5AN1, since they were not observed in cells that do not express OR5AN1 (Mock). On the other hand, Ambrettolide, Phenyl ethyl alcohol (PEA), Lyral®, and Cis-3-hexanol did not cause an OR5AN1 response.
On the other hand, OR1A1 (FIG. 1) showing weaker responsiveness to Muscone than OR5AN1 was also activated by PEA and Cis-3-hexanol (FIG. 4).
It was confirmed that OR10G4 has responsiveness to Guiacol and Ethylvanillin (FIG. 5A).
Regarding OR9Q2, as shown in FIG. 2, responsiveness to p-Cresol, which is a urine odor-causing substance, was confirmed, and responsiveness to p-Creyl Acetate, p-Creyl iso butyrate, and p-Methyl quinoline was further improved. Was found (FIG. 5B). On the other hand, it was found that neither OR10G4 nor OR9Q2 has responsiveness to Muscone (FIG. 5C).
実施例3 匂い物質による交差順応作用の官能評価
1)方法
 実施例2で評価した匂い物質による交差順応作用を、官能試験によって評価した。ガラス瓶(柏洋硝子No.11、容量110mL)に標的匂い物質を加え、標的サンプルとした。標的匂い物質としては、Muscone、Guaiacol又はp-Cresolを用いた。Musconeは、綿球に1mgを染み込ませたものを用いた。Guaiacolとp-Cresolはどちらも、10ppm濃度のミネラルオイル(Sigma-Aldrtich)溶液1mLを用いた。同様の手順で、試験物質をガラス瓶に加え、試験サンプルとした。試験物質としては、5mgのEthylvanillin粉末、又は1000ppmに調製したp-Cresyl Acetate、p-Cresyl iso butyrate、若しくはp-Methyl quinolineのミネラルオイル(Sigma-Aldrtich)溶液1mLを用いた。
 官能試験はパネラー5~8名で単盲方式にて行った。パネラーに、まず標的サンプルを呈示し、その匂い強度を回答させた。次に、試験サンプルを2分間、あるいは匂いが感知できなくなるまで呈示した。その後、改めて標的サンプルを提示し、その匂い強度を評価させた。これを1セットとし、1日に1人あたり4セットを上限として行った。各セット間には少なくとも10分以上の休憩を設けた。
 評価した標的匂いの強度は、両端にそれぞれ「No Odor」、「Strong Odor」と尺度を示した9.5cmの直線上に記入させた。最初の評価での標的匂い強度記入位置とNo Odorとの距離(A)と、2回目の評価での標的匂い強度記入位置とNo Odorとの距離(B)を求め、最初の評価に対する%匂い強度(%Odor intensity of initial estimate)[B/A*100-100]を算出した。この値が低いほど、2回目に嗅いだ標的匂いの強度が1回目に比べて強く抑制された、すなわち強く交差順応したことを表す。この値が-100%である場合、2回目に標的匂いが全く感じられなかったことを意味する。
Example 3 Sensory Evaluation of Cross Adaptation Action by Odor Substance 1) Method The cross adaptation action by the odor substance evaluated in Example 2 was evaluated by a sensory test. A target odor substance was added to a glass bottle (Saiyo Glass No. 11, volume 110 mL) to obtain a target sample. As the target odor substance, Muscone, Guiacol or p-Cresol was used. The Muscone used was a cotton ball soaked with 1 mg. For both Guiacol and p-Cresol, 1 mL of 10 ppm mineral oil (Sigma-Aldrich) solution was used. In the same procedure, a test substance was added to a glass bottle to obtain a test sample. As a test substance, 5 mg of Ethylvanillin powder, or 1 mL of p-Cresyl Acetate, p-Creyl isobutyrate, or p-Methyl quinoline mineral oil (Sigma-Aldrich) solution prepared to 1000 ppm was used.
The sensory test was conducted in a single blind manner by 5 to 8 panelists. The panelist was first presented with the target sample and the odor intensity was answered. The test samples were then presented for 2 minutes or until no odor was detectable. Then, the target sample was again presented and the odor intensity was evaluated. This was set as 1 set, and 4 sets per person per day were set as the upper limit. There was a break of at least 10 minutes between each set.
The intensity of the target odor evaluated was entered on a straight line of 9.5 cm showing the scales of “No Odor” and “Strong Odor” at both ends, respectively. The distance (A) between the target odor intensity entry position and No Odor in the first evaluation and the distance (B) between the target odor intensity entry position and No Odor in the second evaluation are obtained, and the% odor with respect to the first evaluation Intensity (% Oor intensity of initial estimate) [B / A * 100-100] was calculated. The lower this value is, the stronger the intensity of the target odor smelled at the second time is compared with the first time, that is, the stronger the cross adaptation. If this value is −100%, it means that the target odor was not felt at all for the second time.
2)結果
 官能試験の結果を図6に示す。ムスコンに対しては、OR5AN1を活性化した4つの匂い物質はいずれも交差順応を引き起こし、一方、OR5AN1を活性化しなかった4つの匂い物質は全て交差順応を引き起こさなかった(図6A)。特に、低応答性のムスコン受容体であるOR1A1を活性化したPEAとCis-3-hexanolがムスコンに対して交差順応を起こさなかったことから、交差順応を起こすためには比較的高い応答性をもつ受容体を標的とすることが重要であることが判明した。また、Ambrettolideは、ムスコンと良く似た質の匂いを示すのにも関わらず、交差順応を起こさなかった。このことから、匂いの交差順応は匂いの質が認識される高次脳領域よりも末梢側で生じていることが示唆され、匂いの交差順応が嗅覚受容体レベルで説明できるという本発明の原理が支持された。
2) Results The results of the sensory test are shown in FIG. For Muscon, all four odorants that activated OR5AN1 caused cross adaptation, while all four odorants that did not activate OR5AN1 did not cause cross adaptation (FIG. 6A). In particular, PEA and Cis-3-hexanol that activated OR1A1, which is a low-responsive muscone receptor, did not cause cross adaptation to muscone. It turned out to be important to target the receptors with In addition, Ambrettolide did not cause cross-adaptation despite showing a similar odor of Muscon. This suggests that cross-adaptation of odors occurs more peripherally than higher brain regions where odor quality is recognized, and the principle of the present invention that odor cross-adaptation can be explained at the olfactory receptor level Was supported.
 Guaiacolの匂いに対して最も応答性が高い嗅覚受容体はOR10G4である(図1)。官能試験の結果、Guaiacolの匂いは、OR10G4を活性化するEthylvanillinによって交差順応させられることが判明した(図6B)。一方、Guaiacolの匂いは、OR10G4を活性化しないMusconeによって交差順応を起こさず、またEthylvanillinの匂いもMusconeによって交差順応を起こさなかった。この結果より、標的匂いの原因物質に高反応性の嗅覚受容体で認識される別の物質は、該標的匂いの交差順応を起こすという原則がOR10G4に関しても適用できることが示された。 OR10G4 is the olfactory receptor having the highest response to the odor of Guiacol (FIG. 1). As a result of the sensory test, it was found that the odor of Guiacol was cross-adapted by Ethylvanillin that activates OR10G4 (FIG. 6B). On the other hand, the odor of Guiacol was not cross-adapted by Muscone that did not activate OR10G4, and the smell of Ethylvanillin was not cross-adapted by Muscone. From this result, it was shown that the principle that another substance recognized by the olfactory receptor highly reactive to the target odor causative substance causes cross-adaptation of the target odor can also be applied to OR10G4.
 当該原則に普遍性があることを検証するために、p-Cresol-OR9Q2ペアに関してもOR9Q2を活性化する別の匂い物質により交差順応が起こるかを調べた。その結果、p-Cresolの匂いに対しては、p-Cresol受容体OR9Q2を活性化するp-Cresyl Acetate、p-Cresyl iso butyrate、及びp-Methyl quinolineが交差順応を引き起こした(図6C)。一方、OR9Q2を活性化しないMusconeは、p-Cresolの匂いに対しても、p-Cresyl Acetate、p-Cresyl iso butyrate、及びp-Methyl quinolineの匂いに対しても、いずれも交差順応を起こさなかった。 In order to verify the universality of the principle, it was examined whether cross-adaptation occurs with another odorant that activates OR9Q2 for the p-Cresol-OR9Q2 pair. As a result, for the odor of p-Cresol, p-Cresyl Acetate, p-Cresyl isobutyrate, and p-Methyl quinoline that activate the p-Cresol receptor OR9Q2 caused cross adaptation (FIG. 6C). On the other hand, Muscone that does not activate OR9Q2 does not cause cross adaptation to the odor of p-Cresol, nor to the odor of p-Cresyl Acetate, p-Cresyl iso butyrate, and p-Methyl quinoline. It was.
 以上の結果より、匂いの交差順応と、嗅覚受容体の活性化との因果関係が実証され、標的匂いの原因物質に対して高い応答性をもつ嗅覚受容体を活性化し脱感作させられる物質が標的匂いの交差順応を引き起こすという原則が初めて明らかになった。この原則に基づいて、本発明は、嗅覚受容体の応答の活性化を指標として、交差順応に基づいて標的匂いを抑制することができる匂い物質を同定するという新規な方法を提供する。 Based on the above results, a causal relationship between odor cross-adaptation and activation of olfactory receptors has been demonstrated, and substances that activate and desensitize olfactory receptors with high responsiveness to the causative substances of target odors For the first time, the principle that causes cross-adaptation of target odors was revealed. Based on this principle, the present invention provides a novel method for identifying an odorant capable of suppressing a target odor based on cross-adaptation, using activation of an olfactory receptor response as an index.

Claims (22)

  1.  標的の匂いの交差順応を引き起こす物質の選択方法であって、
    (1)嗅覚受容体ポリペプチドを探索し、標的の匂いの原因物質に応答するものを同定すること、
    (2)該同定された嗅覚受容体ポリペプチドに、該標的の匂いの原因物質とは異なる物質である試験物質を添加して、その応答を測定すること、
    (3)該嗅覚受容体ポリペプチドの応答を活性化させた該試験物質を、該標的の匂いの交差順応を引き起こす物質として選択すること、
    を含む、方法。
    A method of selecting a substance that causes cross-adaptation of a target odor,
    (1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent;
    (2) adding a test substance which is a substance different from the target odor causing substance to the identified olfactory receptor polypeptide, and measuring the response;
    (3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
    Including the method.
  2.  前記(1)において、前記嗅覚受容体ポリペプチドが、ヒト嗅覚受容体、及びそれとアミノ酸配列において少なくとも80%同一であって匂い分子の結合によって活性化するポリペプチドである、請求項1記載の方法。 The method according to claim 1, wherein, in (1), the olfactory receptor polypeptide is a human olfactory receptor and a polypeptide that is at least 80% identical in amino acid sequence and activated by the binding of an odor molecule. .
  3.  前記(2)において、前記試験物質を添加する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又は2記載の方法。 In (2), the olfactory receptor polypeptide to which the test substance is added is expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide. 2. The method according to 2.
  4.  前記(2)において、前記嗅覚受容体ポリペプチドの応答が、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、あるいはカルシウムイメージング、又は電気生理学的手法により測定される、請求項1~3のいずれか1項記載の方法。 4. In (2), the response of the olfactory receptor polypeptide is measured by measuring intracellular cAMP amount by ELISA or reporter gene assay, calcium imaging, or electrophysiological technique. The method described in the paragraph.
  5.  (4)官能試験により、前記(3)で選択された物質の中から、前記標的の匂いの交差順応を引き起こす物質を選択すること、
    をさらに含む、請求項1~4のいずれか1項記載の方法。
    (4) selecting a substance that causes cross-adaptation of the target odor from the substances selected in (3) by a sensory test;
    The method according to any one of claims 1 to 4, further comprising:
  6.  前記標的の匂いがタバコ臭である、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, wherein the target odor is a tobacco odor.
  7.  前記標的の匂いが尿臭である、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, wherein the target odor is urine odor.
  8.  標的の匂いを抑制する物質の選択方法であって、
    (1)嗅覚受容体ポリペプチドを探索し、標的の匂いの原因物質に応答するものを同定すること、
    (2)該同定された嗅覚受容体ポリペプチドに、該標的の匂いの原因物質とは異なる物質である試験物質を添加して、その応答を測定すること、
    (3)該嗅覚受容体ポリペプチドの応答を活性化させた該試験物質を、該標的の匂いを抑制する物質として選択すること、
    を含む、方法。
    A method for selecting a substance that suppresses the odor of a target,
    (1) searching for olfactory receptor polypeptides and identifying those that respond to the target odor causative agent;
    (2) adding a test substance which is a substance different from the target odor causing substance to the identified olfactory receptor polypeptide, and measuring the response;
    (3) selecting the test substance that has activated the response of the olfactory receptor polypeptide as a substance that suppresses the odor of the target;
    Including the method.
  9.  前記(1)において、前記嗅覚受容体ポリペプチドが、ヒト嗅覚受容体、及びそれとアミノ酸配列において少なくとも80%同一であって匂い分子の結合によって活性化するポリペプチドである、請求項8記載の方法。 9. The method according to claim 8, wherein in (1), the olfactory receptor polypeptide is a human olfactory receptor and a polypeptide that is at least 80% identical in amino acid sequence and activated by the binding of an odor molecule. .
  10.  前記(2)において、前記試験物質を添加する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8又は9記載の方法。 9. In (2), the olfactory receptor polypeptide to which the test substance is added is expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide. 9. The method according to 9.
  11.  前記(2)において、前記嗅覚受容体ポリペプチドの応答が、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、あるいはカルシウムイメージング、又は電気生理学的手法により測定される、請求項8~10のいずれか1項記載の方法。 The method according to any one of claims 8 to 10, wherein in (2), the response of the olfactory receptor polypeptide is measured by measuring intracellular cAMP amount by ELISA or reporter gene assay, calcium imaging, or electrophysiological technique. The method described in the paragraph.
  12.  (4)官能試験により、前記(3)で選択された物質の中から、前記標的の匂いを抑制する物質を選択すること、
    をさらに含む、請求項8~11のいずれか1項記載の方法。
    (4) selecting a substance that suppresses the odor of the target from the substances selected in (3) above by a sensory test;
    The method according to any one of claims 8 to 11, further comprising:
  13.  前記標的の匂いがタバコ臭である、請求項8~12のいずれか1項記載の方法。 The method according to any one of claims 8 to 12, wherein the target odor is a tobacco odor.
  14.  前記標的の匂いが尿臭である、請求項8~12のいずれか1項記載の方法。 The method according to any one of claims 8 to 12, wherein the target odor is urine odor.
  15.  標的の匂いの交差順応を引き起こす物質の選択方法であって、
    (1)標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドを準備すること、
    (2)該少なくとも1種の嗅覚受容体ポリペプチドに、該標的の匂いの原因物質とは異なる物質である試験物質を添加して、その応答を測定すること、
    (3)該少なくとも1種の嗅覚受容体ポリペプチドのいずれかの応答を活性化させた該試験物質を、該標的の匂いの交差順応を引き起こす物質として選択すること、
    を含み、
    ここで、
     該標的の匂いがタバコ臭であり、かつ該少なくとも1種の嗅覚受容体ポリペプチドが、配列番号4で示されるアミノ酸配列からなるポリペプチド、及び該アミノ酸配列と少なくとも80%同一であってタバコ臭原因物質に対して応答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種であるか、又は
     該標的の匂いが尿臭であり、かつ該少なくとも1種の嗅覚受容体ポリペプチドが、配列番号6で示されるアミノ酸配列からなるポリペプチド、及び該アミノ酸配列と少なくとも80%同一であって尿臭原因物質に対して応答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、
    方法。
    A method of selecting a substance that causes cross-adaptation of a target odor,
    (1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent;
    (2) adding to the at least one olfactory receptor polypeptide a test substance which is a substance different from the target odor causing substance, and measuring the response;
    (3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that causes cross-adaptation of the target odor;
    Including
    here,
    The target odor is tobacco odor, and the at least one olfactory receptor polypeptide is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 and the tobacco odor is at least 80% identical to the amino acid sequence. At least one selected from the group consisting of polypeptides responsive to causative agents, or the target odor is urine odor, and the at least one olfactory receptor polypeptide is a sequence A polypeptide consisting of the amino acid sequence shown by No. 6 and at least one selected from the group consisting of polypeptides that are at least 80% identical to the amino acid sequence and have responsiveness to urine odor-causing substances,
    Method.
  16.  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15記載の方法。 The method according to claim 15, wherein the olfactory receptor polypeptide is expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide.
  17.  前記(2)において、前記嗅覚受容体ポリペプチドの応答が、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、あるいはカルシウムイメージング、又は電気生理学的手法により測定される、請求項15又は16記載の方法。 The method according to claim 15 or 16, wherein in (2), the response of the olfactory receptor polypeptide is measured by measuring intracellular cAMP amount by ELISA or reporter gene assay, calcium imaging, or electrophysiological technique.
  18.  (4)官能試験により、前記(3)で選択された物質の中から、前記標的の匂いに対して交差順応を起こさせる物質を選択すること、
    をさらに含む、請求項15~17のいずれか1項記載の方法。
    (4) selecting a substance that causes cross adaptation to the target odor from the substances selected in (3) by a sensory test;
    The method according to any one of claims 15 to 17, further comprising:
  19.  標的の匂いを抑制する物質の選択方法であって、
    (1)標的の匂いの原因物質に応答する少なくとも1種の嗅覚受容体ポリペプチドを準備すること、
    (2)該少なくとも1種の嗅覚受容体ポリペプチドに、該標的の匂いの原因物質とは異なる物質である試験物質を添加して、その応答を測定すること、
    (3)該少なくとも1種の嗅覚受容体ポリペプチドのいずれかの応答を活性化させた該試験物質を、該標的の匂いを抑制する物質として選択すること、
    を含み、
    ここで、
     該標的の匂いがタバコ臭であり、かつ該少なくとも1種の嗅覚受容体ポリペプチドが、配列番号4で示されるアミノ酸配列からなるポリペプチド、及び該アミノ酸配列と少なくとも80%同一であってタバコ臭原因物質に対して応答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種であるか、又は
     該標的の匂いが尿臭であり、かつ該少なくとも1種の嗅覚受容体ポリペプチドが、配列番号6で示されるアミノ酸配列からなるポリペプチド、及び該アミノ酸配列と少なくとも80%同一であって尿臭原因物質に対して応答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、
    方法。
    A method for selecting a substance that suppresses the odor of a target,
    (1) providing at least one olfactory receptor polypeptide responsive to a target odor causative agent;
    (2) adding to the at least one olfactory receptor polypeptide a test substance which is a substance different from the target odor causing substance, and measuring the response;
    (3) selecting the test substance that has activated the response of any of the at least one olfactory receptor polypeptide as a substance that suppresses the target odor;
    Including
    here,
    The target odor is tobacco odor, and the at least one olfactory receptor polypeptide is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 and the tobacco odor is at least 80% identical to the amino acid sequence. At least one selected from the group consisting of polypeptides responsive to causative agents, or the target odor is urine odor, and the at least one olfactory receptor polypeptide is a sequence A polypeptide consisting of the amino acid sequence shown by No. 6 and at least one selected from the group consisting of polypeptides that are at least 80% identical to the amino acid sequence and have responsiveness to urine odor-causing substances,
    Method.
  20.  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19記載の方法。 20. The method of claim 19, wherein the olfactory receptor polypeptide is expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide.
  21.  前記(2)において、前記嗅覚受容体ポリペプチドの応答が、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、あるいはカルシウムイメージング、又は電気生理学的手法により測定される、請求項19又は20記載の方法。 21. The method according to claim 19 or 20, wherein in (2), the response of the olfactory receptor polypeptide is measured by measuring intracellular cAMP amount by ELISA or reporter gene assay, calcium imaging, or electrophysiological technique.
  22.  (4)官能試験により、前記(3)で選択された物質の中から、前記標的の匂いを抑制する物質を選択すること、
    をさらに含む、請求項19~21のいずれか1項記載の方法。
    (4) selecting a substance that suppresses the odor of the target from the substances selected in (3) above by a sensory test;
    The method according to any one of claims 19 to 21, further comprising:
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