US009057090B2

(12) United States Patent (10) Patent No.: US 9,057,090 B2 Kato et al. (45) Date of Patent: *Jun. 16, 2015

(54) METHOD OF IDENTIFYING ANAGENT FOR Malnic, B., "Searching for the Ligands of Odorant Receptors'. Mol INHIBITING ODOR OF PYRAZINE Neurobiol (2007) 35: 175-181. DERVATIVES Malnic, B. et al., “The human family”. PNAS, Feb. 24, 2004, vol. 101, No. 8, pp. 2584-2589. (71) Applicant: Kao Corporation, Tokyo (JP) International Search Report dated Sep. 7, 2012 for Application No. PCT/JP2012,064862. (72) Inventors: Aya Kato, Tochigi (JP): Naoko Saito, Zhuang, H., et al., “Synergism of Accessory Factors in Functional Utsunomiya (JP); Michiaki Inoue, Expression of Mammalian Odorant Receptors'. Journal of Biologi Cincinnati, OH (US); Kayoko Nomizu, cal Chemistry, vol. 282, No. 20, May 1, 2007, pp. 15284-15293. International Search Report and Written Opinion dated Feb. 13, 2014 Chuo-ku (JP) for Application No. PCT/IB2012/003131. (73) Assignee: KAO CORPORATION, Tokyo (JP) Hindenland, D.M., et al., “Reducing Odiferous Volatiles with Zeolites”. Cosmetics &.Toiletries, vol. 123, No. 7, Jul. 1, 2008, pp. (*) Notice: Subject to any disclaimer, the term of this 67-74. XPO09 167115. patent is extended or adjusted under 35 Kawasaki, K., “Odor masking compositions containing fragrant Sub stances for hair cosmetics', Database Caplus Online Chemical U.S.C. 154(b) by 0 days. Abstracts Service, 2003. XP002719480. This patent is Subject to a terminal dis Shimazaki, K., et al., “Alkylpyrazine-odor-blocking agents contain claimer. ing pyrazines', Database Caplus Online Chemical Abstracts Ser vice, Jan. 28, 2003. XP002719444. (21) Appl. No.: 13/693,295 Shimazaki, K., et al., “Evaluation of the odor activity of pyrazine derivatives using structural and electronic parameters derived from conformational study by molecular mechanics (MM3) and ab initio (22) Filed: Dec. 4, 2012 calculations”, Journal of Molecular Structure, 749 (2005), pp. 169 176. (65) Prior Publication Data Abstract and English Machine Translation of Japanese Application. US 2013/0210O22 A1 Aug. 15, 2013 JP 11-241089. Abstract and English Machine Translation of Japanese Patent JP 2005-325055. Abstract and English Machine Translation of Japanese Patent JP Related U.S. Application Data 2012-050411. (60) Provisional application No. 61/596,967, filed on Feb. Abstract and English Machine Translation of Japanese Patent JP 9, 2012. 2012-050781. Waller, G., and Feather, M. Eds., “The Maillard Reaction in Foods (51) Int. Cl. and Nutrition'. ACS Symposium Series vol. 215, Apr. 29, 1983, GOIN33/53 (2006.01) Chapter 12, pp. 185-286. CI2O I/02 (2006.01) (Continued) GOIN33/566 (2006.01) (52) U.S. Cl. Primary Examiner — Michael Pak CPC ...... CI2O I/025 (2013.01); G0IN33/566 (74) Attorney, Agent, or Firm — Frost Brown Todd LLC (2013.01); G0IN 2500/10 (2013.01) (58) Field of Classification Search (57) ABSTRACT None A method to identify an agent for reducing an odor of a See application file for complete search history. pyrazine derivative, comprising: adding a test Substance and a pyrazine derivative repre (56) References Cited sented by the following Formula (I): U.S. PATENT DOCUMENTS 5,272,134 A 12/1993 Berliner (I) 7,344,845 B2 3/2008 Han et al. 7,585,833 B2 9, 2009 Fadel et al. 2008, OO32913 A1 2/2008 Finke et al. 2 2013/0216492 A1 8, 2013 Kato et al. R3DOC N R2 FOREIGN PATENT DOCUMENTS wherein R represents methyl, ethyl, or acetyl; and R. R. JP 11-241089 9, 1999 and Reach independently represent hydrogen or methyl, to JP A 2003-24423 1, 2003 JP 2005-325055 11, 2005 an olfactory receptor OR5K1 or a polypeptide having at least JP 2012-050411 3, 2012 80% identity to the amino acid sequence of the olfactory JP 2012-050781 3, 2012 receptor OR5K1; WO WO 2005/046632 5, 2005 measuring a response of the olfactory receptor to the pyra WO WO 2012/O29922 3, 2012 Zine derivative; and OTHER PUBLICATIONS identifying a test Substance inhibiting the response of the olfactory receptor on the basis of the measured response. International Search Report dated Mar. 6, 2013 for Application No. PCT/US2012,067889. 9 Claims, 3 Drawing Sheets US 9,057,090 B2 Page 2

(56) References Cited Imahori, K, et al., “Skatole.” Biochemistry dictionary, Mar. 1, 2000, The Fourth Impression of the 3' Ed., pp. 726-727. OTHER PUBLICATIONS Touhara, K., "Function of the sense of Smell receptor—Expression English Abstract and Machine Translation for Japanese Publication and function of the olfactory receptor gene Superfamily.” Igaku No No. JPA 2003-24423, Jan. 28, 2003. Ayumi, Ushiyaku Pub. Inc., Jan. 1, 2005, vol. 212, No. 1, pp. 77-81. Oka, Y, et al., “The identification of the sense of Smell receptor Abstract and English Machine Translation of Japanese Application. antagonistand physiologic significance.” A Collection of Japan Soci ety for Bioscience, Mar. 5, 2003, col. 2003, pp. 152. JP 11-241089, Sep. 9, 1999. Oka, Y, et al., “Olfactory receptor antagonism between odorants.” Abstract and English Machine Translation of Japanese Patent JP The EMBO Journal, 2004, vol. 23, No. 1, pp. 120-126. 2005-325055, Nov. 24, 2005. Saito, H., et al., “Odor coding by a Mammalian receptor repertoire.” Abstract and English Machine Translation of Japanese Patent JP Sci. Signal., 2009, vol. 2, No. 60, ra9, pp. 1-28. Sanz, G., et al., “Comparison of odorant specificity of two human 2012-050411, Mar. 15, 2012. olfactory receptors form different phylogenetic classes and evidence Abstract and English Machine Translation of Japanese Patent JP for antagonism.” Chem. Sense, 2005, vol. 30, No. 1, pp. 69-80. 2012-050781, Mar. 15, 2012. U.S. Patent Jun. 16, 2015 Sheet 1 of 3 US 9,057,090 B2

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US 9,057,090 B2 1. 2 METHOD OF DENTIFYING ANAGENT FOR a malodor, and therefore differs in mechanism from a mal INHIBITING ODOR OF PYRAZINE odor reducing method by adding a perfume, an aromatic, or a DERVATIVES like Substance to the target odorant. In addition, the odorofan aromatic causing an unpleasant sensation to users can be CROSS REFERENCE TO RELATED prevented. Therefore, odor modulation based on olfactory APPLICATION receptor antagonism is a preferred means for reducing mal odor. This application claims priority from U.S. Provisional In order to mask a malodor with an odor of for example, a Patent Application No. 61/596,967 filed on Feb. 9, 2012, perfume or an aromatic, it is necessary to identify an odor which is incorporated herein by reference in its entirety. 10 Substance exhibiting an effective malodor reducing function on the target malodor substance. Hitherto, odors have been FIELD OF THE INVENTION evaluated through a sensory test by specialists. However, the sensory test has problems. The problems include for example, The present invention relates to a method to identify an odor-evaluators must be trained, and the throughput of the test agent for inhibiting an odor of a pyrazine derivative. 15 is low. Thus, identification of an odor Substance exhibiting a malodor reducing function is not easy. BACKGROUND OF THE INVENTION A skin tanning agent (also called a self-tanning agent or a Sunless tanning agent) is a skin cosmetic for staining the skin. There are a large number of malodorous molecules having As a component for providing skin with a brown color, dihy different polarities and molecular weights in our living envi droxyacetone (DHA) is mainly used alone or together with, ronment. Hitherto, a variety of methods have been developed for example, erythrulose. These components react with the for reducing various malodors. In general, the methods for upper layer of the skin to change the color of skin to brown. reducing malodors are roughly classified into biological This staining probably progresses by a browning reaction, but methods, chemical methods, physical methods, and sensory the details of the reaction mechanism have been hardly clari methods. Among malodorous molecules, short-chain fatty 25 fied. The browning reaction is also called a Maillard reaction acids and amines, having high polarity, can be reduced by in the food chemistry field and is a term referring to a reaction chemical methods, i.e., neutralization. Sulfur compounds for generating a brown polymer called melanoidin by poly Such as thiol can be deodorized by physical methods, i.e., merizing a nitrogen-containing compound. Such as an amino absorptive treatment. However, there still remain many mal acid or a , and a reducing Sugar. The Maillard reaction odorous molecules which cannot be reduced by known mal 30 is caused by, for example, heating of a food and is involved in odor reducing methods. coloring of the food and generation of a flavor component. In addition, the known malodor reducing methods by the chemical neutralization or the absorptive treatment take long Pyrazines are known to be generated by the Maillard reac time for reducing the amounts of malodorous Substances and tion and as a component of a roasted aroma of a food. Patent thereby lack immediate effect. A method of reducing malodor 35 Document 1 describes an odor-blocking agent including a by using an aromatic and thereby providing strong perception functional pyrazine compound as an active ingredient for of another odor is also known. However, the odor of the blocking the odor of an alkylpyrazine compound. Pyrazines aromatic may cause discomfort in this method. Moreover, are usually used as flavoring agents for foods, and the odors of these known methods may get rid of odors other than the pyrazines are rather utilized. In the food field, odors obtained target malodors. Thus, there is a demand for a malodor reduc 40 by the Maillard reaction are reported in Non-Patent Docu ing method which can overcome these problems. ment 1. In Non-Patent Document 1, the pyrazine compound is In mammals including humans, the mechanism for odorant described as a preferred odor which characterizes a food. For recognition includes binding odorant molecules to olfactory example, 2,6-dimethylpyrazine is described as having a receptors present on olfactory sensory neurons included in “sweet, fried, resembling fried potatoes, nutty, roasted odor. the olfactory epithelium, which is present in an upper portion 45 of the nasal cavity, and transmitting the response of the recep PRIOR ART tors to the central nervous system. It has been reported that, 387 different olfactory receptors are present in human, and the encoding these olfactory receptors account for Patent Document about 3% of the . In general, a plurality of 50 olfactory receptors responds to a plurality of odorant mol Patent Document 1 JP-A-2003-024423 ecules. Specifically, one single olfactory receptor responds to a plurality of structurally similar odorant molecules at differ Non-Patent Document ent affinities, while one single odorant molecule is detected by a plurality of olfactory receptors. It is also reported that a 55 Non-Patent Document 1 S. Fors. Sensory Properties of certain odorant molecule which can activate one olfactory Volatile Maillard Reaction Products and Related Com receptor serves as an antagonist which inhibits activation of pounds, “The Maillard Reaction in Foods and Nutrition' another olfactory receptor. Such combined response of these (ACS Symposium Series, Vol. 215), Chapter 12, pp. 185 olfactory receptors leads to recognition of each odor. 286 Publication Date (Print): Apr. 29, 1983 Thus, when a first odorant molecule is co-present with a 60 second odorant molecule, in Some cases, the response of an olfactory receptor to the first odorant molecule is inhibited by SUMMARY OF THE INVENTION the second odorant molecule. Through the inhibition, the odor of the first odorant molecule recognized by olfactory The present invention provides a method to identify an receptors may vary considerably. This mechanism is called 65 agent for reducing an odorofapyrazine derivative, including: “olfactory receptor antagonism.” Odor modulation by olfac adding a test Substance and a pyrazine derivative repre tory receptor antagonism can inhibits recognition specific to sented by the following Formula (I): US 9,057,090 B2 4 As used herein, the term “masking in the odor-related (I) field generally refers to means for reducing or weakening recognition of a target odor. The term “masking may encom pass chemical means, physical means, biological means, and sensory means. Examples of the masking means include any means for removing an odorant molecule responsible for a target odor from the environment (e.g., adsorption and chemi wherein R represents methyl, ethyl, or acetyl; and R. R. cal decomposition of the odorant); means for preventing and Reach independently represent hydrogen or methyl, to release of a target odor to the environment (e.g., sealing); and olfactory receptor OR5K1 or a polypeptide having at least 10 a method in which recognition of a target odor is reduced by 80% identity to the amino acid sequence of the olfactory adding another odorant Such as a flavoring agent or an aro receptor OR5K1; matic. measuring a response of the olfactory receptor to the pyra As used herein, the term “masking through olfactory recep Zine derivative; and 15 tor antagonism” refers to one embodiment of the aforemen identifying a test Substance inhibiting the response of the tioned broadly defined “masking and is means for inhibiting olfactory receptor on the basis of the measured response. the response of an olfactory receptor to a target odorant mol ecule by an additional odorant molecule, to thereby modulate the odor of the target odorant molecule recognized by a Sub BRIEF DESCRIPTION OF THE DRAWINGS ject. Although masking through olfactory receptor antago FIG. 1 shows responses of olfactory receptor OR5K1 to nism employs an additional odorant molecule, the masking differs from means for canceling out a target odor by use of a various concentrations of 2,6-dimethylpyrazine, wherein n=5 strong odorant such as a perfume. In one embodiment of and error bar-tSE; masking through olfactory receptor antagonism, a Substance FIG. 2 shows responses of olfactory receptor OR5K1 to which can inhibit the response of an olfactory receptor Such as various concentrations of pyrazine derivatives, wherein n=3 25 an antagonist is used. When a response-inhibiting Substance to 5 and error bar-tSE; and which can specifically inhibit the response of a receptor FIG.3 shows effects of various compounds for reducing an related to recognition of a certain odor is employed, the odor of 2,6-dimethylpyrazine, wherein error bar-ESE. response of the receptor is Suppressed, whereby the odor DETAILED DESCRIPTION OF THE INVENTION 30 recognized by a subject can be modulated. “Pyrazine derivatives” in the present invention are com A problem that the use of a commercially available skin pounds represented by the following Formula (I): tanning agent (self-tanning agent or Sunless tanning agent) involves a characteristic unpleasant odor expressed as, for example, an earthy or burnt Sugar odor has been reported (D. 35 (I) M. Hindenlang and M. E. McDonnell, Cosmetics & Toiletries magazine, 2008, Vol. 123, No. 7, pp. 67-74), and there has 2 been a demand for improvement thereof. The cause of the R3 CN R2 unpleasant odor has been investigated to reveal that dimeth ylpyrazine, which is generated when dihydroxyacetone 40 (DHA) or erythrulose in a skin tanning agent reacts with the In Formula (I), R represents methyl, ethyl, or acetyl; and skin to change the color of skin to brown, is one of causative R. R. and R. each independently represent hydrogen or substances. Furthermore, it has been revealed that, in addition methyl. At least one of R to R is preferably an alkyl group. to dimethylpyrazine, other pyrazine derivatives yield similar Examples of the pyrazine represented by Formula (I) of the odors, and there is a demand for reducing the odor of these 45 present invention include 2,6-dimethylpyrazine, 2,5-dimeth pyrazine derivatives. ylpyrazine, 2,3-dimethylpyrazine, 2,3,5-trimethylpyrazine, In order to attain the olfactory receptor antagonism, an 2,3,5,6-tetramethylpyrazine, 2-monomethylpyrazine, 2-mo olfactory receptor which responds to a target malodorous noethylpyrazine, 2-ethyl-6-methylpyrazine, 3-methyl-2- substance must be determined, and substance which effec acetylpyrazine, and 3.5-dimethyl-2-acetylpyrazine. tively exhibits an antagonistic effect on an olfactory receptor 50 In the present invention, the “odorofapyrazine derivative' of malodorous Substance must be searched and identified. can be an odor due to a pyrazine derivative represented by Accordingly, the present inventors have searched for an olfac Formula (I). The “odorofapyrazine derivative' in the present tory receptor which responds to a pyrazine derivative and invention can be an odor generated by a Maillard reaction and have succeeded in identification of the receptor. Furthermore, is typically described as a roasted odor and the like. For the present inventors have found that the substance which 55 example, Non-Patent Document 1 describes that 2,6-dimeth inhibits response of the olfactory receptor can reduce the odor ylpyrazine has a “Sweet, fried, resembling fried potatoes, of the pyrazine derivative through masking by the olfactory nutty, roasted odor. In addition, the “odor of a pyrazine receptor antagonism. derivative' in the present invention can be expressed as an According to the present invention, an agent for reducing odor generated when a known self-tanning agent is applied to the odor of a pyrazine derivative can be efficiently identified. 60 skin, more specifically, an earthy odor or an odor generated According to an inhibitor identified by the present invention, when dihydroxyacetone(DHA) or erythrulose reacts with the the odor of a pyrazine derivative, for example, the odor gen skin to change the color of skin to brown. erated by using a known self-tanning agent (or Sunless tan The present inventors identified olfactory receptor OR5K1 ning agent) can be specifically reduced without causing prob from many olfactory receptors as a sole receptor which lems which occur in known malodor reducing methods using 65 responds to pyrazine derivatives. The response of OR5K1 to a deodorant or an aromatic, such as lack of immediate effect pyrazine derivatives has not been found until now, and and discomfort due to an odor of an aromatic. OR5K1 is a novel receptor for the pyrazine derivatives. US 9,057,090 B2 5 6 As shown in FIGS. 1 and 2, OR5K1 responds to pyrazine neered recombinant cells expressing the olfactory receptor, or derivatives represented by Formula (I) in dose-dependent a culture of such cells can be used. The recombinant cells can manner. Consequently, a Substance which inhibits the be produced by transforming cells with a vector carrying a response of OR5K1 causes a change in perception of odors of gene encoding the olfactory receptor. On this occasion, pyrazine derivatives in the central nervous system by masking 5 RTP1S is preferably transfected together with the receptor in based on the olfactory receptor antagonism and, as a result, order to enhance expression of the olfactory receptor in cell can reduce the odor in an odor-specific manner. membrane. Accordingly, an aspect of the present invention provides a Examples of the RTP1S which can be used in the produc method to identify an agent for reducing an odorofapyrazine tion of the recombinant cells include human RTP1 S. Human derivative. The method includes the steps of adding a test 10 RTP1S has been registered in GenBank under the accession Substance and a pyrazine derivative represented by Formula number GI: 50234917. Alternatively, a polypeptide having an (I) to olfactory receptor OR5K1; measuring response of the amino acid sequence which has at least 80%, preferably at olfactory receptor to the pyrazine derivative; and identifying least 85%, more preferably at least 90%, more preferably at a test Substance inhibiting the response of the olfactory recep least 95%, and more preferably at least 98% identity to human tor on the basis of the measured response. The identified test 15 RTP1S and, like human RTP1S, enhancing expression of the Substance is selected as the agent for reducing an odor of a olfactory receptor in membrane may be used instead of pyrazine derivative. The method of the present invention can human RTP1 S. For example, an RTP1S variant used in be performed in vitro or ex vivo. Examples described in this specification is a protein encoded In the method of the present invention, a test Substance and by a gene having a nucleotide sequence represented by SEQ a pyrazine derivative serving as a causative Substance for an ID NO:3 and having the amino acid sequence represented by odor are added to olfactory receptor OR5K1. The pyrazine SEQ ID NO: 4. The protein has an amino acid sequence derivative used in the method of the present invention may be having 78.9% identity to human RTP1S, enhances expression any pyrazine derivative represented by Formula (I). In the of the olfactory receptor in membrane, and can be used in pyrazine derivative represented by Formula (I) used in the production of the recombinant cells. Alternatively, mouse method of the present invention, at least one of R to R is 25 RTP1S (see Sci. Signal., 2009, 2060): ragmentioned above) is preferably an alkyl group. also a protein which has an amino acid sequence having 89% Preferred examples of the pyrazine derivative used in the identity to human RTP1S, enhances expression of the olfac method of the present invention include 2,6-dimethylpyra tory receptor in membrane, and can be used in production of zine, 2,5-dimethylpyrazine, 2,3-dimethylpyrazine, 2,3,5-tri the recombinant cells. methylpyrazine, 2,3,5,6-tetramethylpyrazine, 2-monometh 30 In this specification, the sequence identity in nucleotide ylpyrazine, 2-monoethylpyrazine, 2-ethyl-6-methylpyrazine, sequence or amino acid sequence is calculated by a Lipman 3-methyl-2-acetylpyrazine, and 3,5-dimethyl-2-acetylpyra Pearson method (Science, 227, 1435, (1985)). Specifically, Z10. the sequence identity is calculated by analysis using a homol Among them, 2-monomethylpyrazine, 2-monoethylpyra ogy analysis (Search homology) program in gene information Zine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 2,6-dim 35 processing software Genetyx-Win (Ver, 5.1.1: Software ethylpyrazine, 2,3,5-trimethylpyrazine, and 2,3,5,6-tetram Development Co., Ltd.) and setting Unit size to compare ethylpyrazine are more preferred; and 2,3-dimethylpyrazine, (ktup) to 2. 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimeth According to the present invention, after addition of a test ylpyrazine, and 2,3,5,6-tetramethylpyrazine are even more Substance and a pyrazine derivative, the response of olfactory preferred. 40 receptor OR5K1 to the pyrazine derivative is measured. The The test substance used in the method of the present inven response of the olfactory receptor may be measured by any tion may be any material which is desired to be used as an method which is known in the art, such as measurement of agent for reducing an odor of a pyrazine derivative. The test intracellular cAMP levels or reporter gene assay. For Substance may be a natural material or may be artificially example, it is known that the olfactory receptor activated by synthesized by a chemical or biological method and may be a 45 an odorant molecule couples with intracellular Gas to activate compound, a composition, or a mixture thereof. adenylate cyclase and thereby increases the amount of intra Olfactory receptor OR5K1 used in the method of the cellular cAMP (Mombaerts P., Nat. Neurosci., 5, pp. 263 present invention is expressed in human olfactory cells and 278). Accordingly, the intracellular cAMP amount after the has been registered in GenBank under the accession number addition of the odorant molecules can be used as an index for GI: 115270955. OR5K1 is a protein encoded by a genehaving 50 measurement of response of the olfactory receptor. The a nucleotide sequence represented by SEQ ID NO: 1 and cAMP amount can be measured by, for example, ELISA or having the amino acid sequence represented by SEQID NO: calcium imaging method. 2. Subsequently, the inhibition effect of a test substance on In the method of the present invention, the olfactory recep the response to the pyrazine derivative is evaluated based on tor can be used in any form that does not lose the responsive 55 the measured response of the olfactory receptor, and thereby ness to a pyrazine derivative. For example, the olfactory a test substance inhibiting the response is identified. The receptor can be used in a form of a tissue, a cell, or a culture inhibition effect can be evaluated by, for example, compari which naturally expresses the olfactory receptor, such as an son of the measured responses of the olfactory receptor to the olfactory organ or olfactory cells isolated from a living body; pyrazine derivative under addition of different concentrations membrane of olfactory cells carrying the olfactory receptor, a 60 of a test substance. More specifically, the response of the recombinant cell genetically engineered to express the olfac olfactory receptor to the pyrazine derivative is compared tory receptor or a culture of the cell; membrane of the recom between a group of high concentration test Substance and a binant cells; or artificial lipid bilayer membrane having the group of low concentration test Substance; between a group olfactory receptor. These forms are included in the range of containing a test Substance and a group not containing the test the olfactory receptor used in the present invention. 65 substance; or between before and after addition of a test In a preferred embodiment, cells, such as olfactory cells, substance. If the response of the olfactory receptor is inhib naturally expressing the olfactory receptor, genetically engi ited by addition of a test substance or by addition of a higher US 9,057,090 B2 7 8 concentration of a test Substance, the test Substance can be utilizing a browning reaction. The pyrazine derivatives may identified as a material which inhibits response of the olfac be contained in foods as flavoring components and may cause tory receptor to the pyrazine derivative. off-flavors if their amounts are high. Accordingly, other In the method of the present invention, a polypeptide hav examples of the compounds or the compositions desired to ing a function equivalent to OR5K1 can be used as an olfac reduce the odor of the pyrazine derivatives include foods tory receptor instead of OR5K1. Examples of the polypeptide containing the pyrazine derivatives as flavoring components include polypeptides composed of amino acid sequences hav and compositions thereof, more specifically, foods, such as ing at least 80%, preferably at least 85%, more preferably at roasted peanuts and milk powders, of which taste is reduced least 90%, more preferably at least 95%, and more preferably by the presence of excess amounts of pyrazine derivatives and at least 98% identity to the amino acid sequence (SEQID NO: 10 2) of OR5K1 and having responsiveness to the pyrazine compositions thereof. derivative. EXAMPLES In the method of the present invention, the above-described OR5K1 and the polypeptides having a function equivalent to The present invention will now be described more specifi that of OR5K1 may be used alone or in a combination of two 15 or more thereofas the olfactory receptor. cally by examples. The test substance identified by the above-described pro Example 1 cedure is a substance which inhibits response of the olfactory receptor to a pyrazine derivative and thereby causes a change Identification of Olfactory Receptor Responding to in perception of the odor of the pyrazine derivative in the central nervous system by masking based on the olfactory Pyrazine Derivative receptor antagonism. As a result, the test Substance can 1) Cloning of Human Olfactory Receptor Gene reduce the level of human perception of the odor of the Human olfactory receptors were cloned based on sequence pyrazine derivative. Consequently, the test Substance identi information registered in GenBank by PCR using human fied by the above-described procedure is selected as an agent 25 genomic DNA female (G1521: Promega Corporation) as a for reducing the odor of the pyrazine derivative. template. Each gene amplified by PCR was inserted into a For example, if the response of the receptor in a group pENTR vector (Invitrogen Inc.) in accordance with the containing a test Substance in the above-described measure manual and was recombined into the Not and AscI site ment procedure is reduced to 60% or less, preferably 50% or located downstream of a Flag-Rho tag sequence inapME 18S less, or more preferably 25% or less of the response in a 30 vector using the NotI and AscI site present in the pENTR control group, the test Substance can be selected as an agent Vector. for reducing the odor of the pyrazine derivative. 2) Production of pME18S-RTP1S Vector The substance selected by the method of the present inven An RTP1S variant gene (SEQ ID NO: 3) encoding an tion can reduce an odor of a pyrazine derivative by olfactory RTP1S variant (SEQID NO: 4) was inserted into the EcoRI masking based on inhibition of response of the olfactory 35 and XhoI site of a pME 18S vector. receptor to the odor of the pyrazine derivative. Consequently, 3) Production of Olfactory Receptor-Expressing Cell the Substance can be used as an active ingredient for reducing HEK293 cells expressing 373 types of human olfactory an odor of a pyrazine derivative, for example, a roasted odor, receptors were produced. A reaction solution having a com an odor generated by a Maillard reaction, an odor generated position shown in Table 1 was prepared and left to stand in a when a self-tanning agent is applied to the skin (e.g., earthy 40 clean bench for 15 min and then was dispensed in each well of odor), or an odor generated when DHA or erythrulose reacts a 96-well plate (Becton, Dickinson and Company). Subse with the skin to change the color of skin to brown. quently, HEK293 cells (100 uL, 3x10 cells/cm) were For example, the substance selected by the method of the seeded in each well and cultured in an incubator at 37° C. and present invention can be an active ingredient of an agent for 5% CO, for 24 hr. reducing an odor of a pyrazine derivative. Alternatively, the 45 substance selected by the method of the present invention can be contained in compounds or compositions for reducing the TABLE 1 odor of pyrazine derivatives as active ingredients for reducing OPTI-MEM (GIBCO) 50 L Human olfactory receptor gene 0.075 ug the odor of the pyrazine derivatives. Alternatively, the antago (incorporated in a pME18S vector having a nists can be used for producing agents for reducing the odor of 50 Flag-Rho tag at the N-terminal) pyrazine derivatives or for producing compounds or compo pGL4.29 (fluc2P-CRE-hygro, Promega Corp.) 0.03 Ig sitions for reducing the odor of pyrazine derivatives. pGL4.75 (hRluc-CMV, Promega Corp.) 0.03 Ig For example, the substance selected by the method of the pME18S-RTP1S variant vector 0.03 Ig present invention can be used as active ingredients for reduc Lipofectamine 2000 (Invitrogen Inc.) 0.4 Il ing the odor of pyrazine derivatives in every compound or 55 composition desired to reduce the odor of the pyrazine deriva 4) Luciferase Assay tives or under every environment desired to reduce the odor of An olfactory receptor expressed in HEK293 cells couples the pyrazine derivatives. Alternatively, the substance selected with endogenous Gas in the cells to activate adenylate cyclase by the method of the present invention can be used for pro and thereby increases the amount of intracellular cAMP. In ducing compounds or compositions desired to reduce the 60 this study, the response of a pyrazine derivative was measured odor of pyrazine derivatives as active ingredients for reducing by luciferase reporter gene assay which monitors an increase the odor of the pyrazine derivatives. Examples of the com in the amount of intracellular cAMP as the luminescence pounds or the compositions desired to reduce the odor of the value derived from a firefly luciferase gene (fluc2P-CRE pyrazine derivatives include skin tanning agents (also called hygro). A Renilla luciferase gene was fused downstream of a self-tanning agent or Sunless tanning agent), for example, a 65 CMV promoter (hPluc-CMV) and was also introduced to skin tanning agent containing dihydroxyacetone (DHA) or HEK293 cells as an internal standard to correct errors in erythrulose as a color former and another skin tanning agent transgenic efficiency and number of cells. US 9,057,090 B2 9 10 The culture medium was removed from the culture pro 2,6-dimethylpyrazine from the luminescence value (X) duced in the above 3), and 75 L of a solution prepared with derived from firefly luciferase induced by stimulation with a CD293 medium (Invitrogen Inc.) So as to contain a pyrazine 2,6-dimethylpyrazine alone. Similarly, the receptor activity derivative (1 mM 2,6-dimethylpyrazine) was added thereto. (Z-Y) in the presence of a test substance was determined by The cells were cultured in a CO, incubator for 2.5 hr to Subtracting the luminescence value (Y) in cells not stimulated sufficiently express the luciferase gene in the cells. The with 2,6-dimethylpyrazine from the luminescence value (Z) luciferase activity was measured with a Dual-GloTM stimulated with a mixture of 2,6-dimethylpyrazine and the luciferase assay system (Promega Corporation) in accor test substance. The reduction rate of the receptor activity dance with the operating manual of the system. The fold (Z-Y) in the presence of a test substance to the receptor increases were calculated by dividing the luminescence value 10 activity (X-Y) by stimulation with 2,6-dimethylpyrazine derived from firefly luciferase induced by stimulation with a alone was calculated by the following computation expres pyrazine derivative at each concentration by the lumines S1O. cence value in cells not stimulated with the pyrazine deriva Inhibition rate (%)={1-(Z-Y)/(X-Y)x100, tive and were used as an index of response strength. 5) Results 15 to determine the receptor response-inhibiting rate of the test The response of each of the 373 types of the olfactory Substance. In the measurement, multiple independent experi receptors to 2,6-dimethylpyrazine (1 mM) was measured, and ments were performed in duplicate, and the average of each the result showed that only olfactory receptor OR5K1 experiment was used. responded to 2,6-dimethylpyrazine (FIG. 1). Response of As shown in Table 2, the results showed that 56 test Sub OR5K1 to 2,6-dimethylpyrazine has not been reported until stances had antagonistic activity on the response of OR5K1 to now, and therefore OR5K1 is a novel 2,6-dimethylpyrazine 2,6-dimethylpyrazine. receptor. TABLE 2

Example 2 Inhibition rate: 25 75% or more Inhibition rate: Response Characteristics of OR5K1 to Pyrazine (exceptionally 50% or more Inhibition rate: Derivatives (strong 40% or more antagonist) (weak antagonist) Olfactory receptor OR5K1 (SEQID NO:2) was expressed 2-ethyl-4-(2,2,3- 2-methoxy-1- cyclo in HEK293 cells together with an RTP1S variant (SEQ ID 30 trimethyl-3- (phenylmethoxy)- pentadecanolide NO: 4) by the same procedure as in Example 1, and the cyclopenten-1- 4-(1-propenyl)- (pentallide) yl)-2-buten-1-ol benzene (benzyl 1-(2,6,6- dependency of the response on the concentration (0, 3, 10.30, (bacdanol) isoeugenol) trimethyl-1,3- 100, 300, and 1000 uM) of various pyrazine compounds was 5-methyl-2-(1- C-methyl-f-(p- cyclohexadien-1- investigated. The pyrazine compounds used in this Example methylethyl)- tert yl)-2-buten-1-one 35 phenol (thymol) butylphenyl)- (damascenone) were pyrazine and pyrazine derivatives selected from the propionaldehyde 2,4-dimethyl-3- group consisting of 2-methylpyrazine, 2,3-dimethylpyrazine, (lilial) cyclohexene-1- 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tet l(-)-menthol carboxyaldehyde ramethylpyrazine and 2-ethylpyrazine. phenylethyl ()-6- (triplal) Salicylate hexadecenelactone isocyclocitral The results showed that OR5K1 responded in a dose-de -(5,6,7,8- (ambrettolide) 6,10-dimethyl-3- pendent manner to the pyrazine derivatives, i.e., 2-meth 40 etrahydro 2-(2-(4-methyl)- oxa-9-undecenal ylpyrazine (2-MMP), 2,3-dimethylpyrazine (2,3-DMP), 2.5- 3,5,5,6,8,8- 3-cyclohexen-1- (citronellyloxy dimethylpyrazine (2,5-DMP), 2,6-dimethylpyrazine hexamethyl-2- yl)-propyl acetaldehyde) naphthalenyl)- cyclopentanone dimethyl benzyl (2,6-DMP), 2,3,5-trimethylpyrazine (2,3,5-TMP), 2,3,5,6- ethanone (Tonalid (nectary) carbinyl acetate tetramethylpyrazine (2,3,5,6-TMP), and 2-ethylpyrazine (registered formaldehyde ethyl (2-MEP), but did not respond to pyrazine (FIG. 2). 45 trademark), cyclododecyl (3aC.4C.7C,3aC.)- entarome) ethyl acetal octahydro-4,7- Example 3 isolongifolanone (Boisambrene methano-3aH 4-(2,6,6- Forte (registered indene-3a trimethyl-2- trademark)) carboxylate Identification of Antagonist of OR5K1 cyclohexen-1-yl)- 1-(2,3,4,7,8,8a (Fruitate 50 3-buten-2-one (C- hexahydro (registered Antagonistic activities of 174 test Substances against olfac ionone) 36,8,8- trademark)) 4-(2,6,6- tetramethyl-1H 2-pentyl-3-methyl tory receptor OR5K1 were investigated by examining trimethyl-1- 3a,7- 2-cyclopenten-1- response of OR5K1 to a pyrazine derivative. cyclohexen-1-yl)- methanoaZulen-5- Ole 2,6-Dimethylpyrazine (1 mM) and a test substance (300 3-buten-2-one (B- yl)-ethanone (dihydrolasmon) 55 ionone) (acetyl cedrene) phenylhexanol uM) shown in Table 2 were added to HEK293 cells express vetiverol B-methyl naphthyl 1-(5,5-dimethyl-1- ing olfactory receptor OR5K1 by the same procedure as in 7-acetyl ketone cyclohexen-1-yl)- Example 2, and the response of the olfactory receptor to 1,2,3,4,5,6,7,8- cedryl acetate 4-penten-1-one 2,6-dimethylpyrazine was measured to evaluate a change in octahydro-1,1,6,7- (5E)-3- (Dynascone response of the receptor due to addition of the test substance. tetramethyl methylcyclopenta (registered naphthalene (Iso E 5-decen-1-one (6- trademark)) Flavoring agents recognized to have cytotoxicity were re 60 Super) muscenone) 6-tert-butyl-1,1- evaluated using a mixture of 333 uM of 2,6-dimethylpyrazine C-amylcinnamic cinnamaldehyde dimethylinden-4-yl and 100 uM of a test substance. aldehyde 4-(1-ethoxyvinyl)- methyl ketone C-methyl-4-(1- 3,3,5,5- (celestolide) The receptor response-inhibiting rate of a test Substance methylethyl)- tetramethyl cis-4-isopropyl was calculated as follows. The receptor activity (X-Y) by benzenepropanol cyclohexanone cyclohexylmethanol stimulation with 2,6-dimethylpyrazine alone was determined 65 (cyclamen (kephalis) (mayol) by subtracting the luminescence value (Y) in cells to which aldehyde) C-hexylcinnamic 1-allyl-3-methoxy the receptor was introduced but were not stimulated with US 9,057,090 B2 11 12 TABLE 2-continued Example 4 Inhibition rate: Evaluation of Ability of Antagonist of OR5K1 for 75% or more Inhibition rate: Reducing the Odor of Pyrazine Derivatives (exceptionally 50% or more inhibition rate: 5 Minist As Rais) The ability to reduce an odorofapyrazine derivative by the antagonist materials identified in Example 3 was confirmed styl-3-deen tly, thement by a sensory test. (undecavertol)-O presidehyde-low- (960,7-dimethyl- A flavoring agent (0.5 LL) WaS dded to dough (0.5 g) 1-(2-tert-butyl- (hydratropic 2,6-octadien-1-ol containing 2,6-dimethylpyrazine (1%, and the odor thereof cyclohexyloxy)-2- aldehyde) (geraniol) was evaluated by comparing the strength of odor of 2.6- butanol (Amber 3,7-dimethyl-6- 4-tert-butyl-2,6- dimethylpyrazine in dough not applied with flavor by the Core (registered octenal dimethyl-3,5- flavoring agent. The sensory evaluation test was performed by trademark)) (citronellal) dinitro- four panelists. In the evaluation, a case where a strong odor of tyle R 2,6-dimethylpyrazine was smelled was rated as 1, and a case tetramethyl-y 1,4-di4-dioxacyclo lo- 15 where theodor of 2,6-dimethylpyrazine was not smelled at all naphtho2,1- heptadecane-5,17- was rated as 5. - - - - - bifuran (Ambrotech dione (ethylene As a result, the odor of 2,6-dimethylpyrazine was inhibited (registered brassylate) by the antagonists which were revealed in Example 3 to trademark)) hexyl salicylate inhibit the response of OR5K1 to 2,6-dimethylpyrazine, that 4,7,7-trimethyl- C.-methyl-3,4- 20 is, the odor of 2,6-dimethylpyrazine was inhibited by 4-(2,6, spirobicyclo[2.2.1 methylenedioxy- 6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one (B-ionone), heptane-2,1- hydrocinnamic 4-methyl-3-decen-5-ol (undecavertol), 1-(2-tert-butylcyclo cyclopentan-3-one aldehyde hexyloxy)-2-butanol (Amber Core (registered trademark)), (registered(Sagetone (helional) C.-methyl-3-(p-tert-butylphenyl)-propionaldehydey p-tert-butylphenyl)-propionaldehyde (lilial(lilial), trademark) V) 2s f-methyl naphthyl ketone, (5E)-3-methylcyclopenta-5-de C-isomethyl ionone cen-1-one (Ö-muscenone), C-hexylcinnamic aldehyde, (Y-methyl ionone) C-isomethyl ionone (Y-methyl ionone), 2-methyl-4-(2,2,3- 3,7-dimethyl-1- trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol (Sandalmysore octanol Core (registered trademark)), Y-undecalactone, 1-(2,6,6-tri Senio so methyl-1,3-cyclohexadien-1-yl)-2-buten-1-one (dama cedryl methyl ether scenone), 2,4-dimethyl-3-cyclohexene-1-carboxyaldehyde SCOC (triplal), ethyl-(3ao,4C,7O.3ao)-octahydro-4,7-methano 2-methyl-4-(2,2,3- 3ah-indene-3a-carboxylate (Fruitate (registered trade trimethyl-3- mark)), 1-(5.5-dimethyl-1-cyclohexen-1-yl)-4-penten-1-one cyclopenten-1-yl)- s (Dynascone (registered trademark)), (E)-3,7-dimethyl-2,6- 2-buten-1-ol(Sandalmysore Core octadien-1 -ol (geraniol), and c-methyl-3,4-methylenedioxy (registered hydrocinnamic aldehyde (helional) (FIG. 3). trademark)) The embodiments of the present invention have been 2- described above, but it should be understood that the specific cyclohexylpropanal embodiments described above are not intended to limit the Od present invention. Various other changes and modifications trademark) II) within the scope of the present invention are obvious to those Y-undecalactone skilled in the art. The literatures and patent applications cited in this speci fication are incorporated by reference herein in their entirety.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 4

<21 Os SEQ ID NO 1 &211s LENGTH: 927 &212s. TYPE: DNA <213> ORGANISM: Homo Sapiens 22 Os. FEATURE: 223 OTHER INFORMATION: ORSK1

<4 OOs SEQUENCE: 1

atggctgaag aaaat catac catgaaaaat gag tittatcc to acaggatt tacagat cac 60

Cctgagctga agactictgct gtttgttggtg ttctittgcca totatctgat Caccgtggtg 12O

gggaat atta gtttggtggc actgatattt acacaccgt.c ggctt cacac accalatgtac 18O

atc.tttctgg gaaatctggc ticttgttggat tcttgctgtg cctdtgctat tacc cc caaa 24 O

atgttagaga act tott titc. tgagaacaaa aggattt coc totatgaatg tgcagtacag 3 OO US 9,057,090 B2 13 - Continued titt tattt to tittgcactgt ggaaactgca gactgct titc ttctggcagc aatggcctat 360 gaccgctatg tggccatatg caa.cccactg cagtaccaca t catgatgtc caagaaactic tgcatt caga tgaccacagg ggc ctitcata gctggaalacc tgcattc cat gatt catgta gggcttgt at ttaggittagt tittctgtgga tcqaatcaca toalaccactt. ttact.gtgat 54 O attict tcc ct tgtatagact citc.ttgttgtt gatcc titata t caatgaact ggttctatt c atctitcticag gttcagttca agt ctittacc atagg tagtg totta at at C titat ct citat 660 at tott citta Ctattittcaa. aatgaaatcc aaagagggaa gggccaaagc ttitt to tacic 72 O tgtgcatc cc acttitttgtc agttt catta ttctatoggat ctottt tott catgitacgitt agaccalaatt tgcttgaaga aggggataaa gatataccag ctgcaattitt atttacalata 84 O gtagttcc ct tactaaat CC titt cattt at agcctgagaa at agggaagt aataagtgtc 9 OO ttaagaaaaa ttctgatgaa gaaataa 927

SEQ ID NO 2 LENGTH: 3O8 TYPE : PRT ORGANISM: Homo Sapines FEATURE: OTHER INFORMATION: ORSK1

SEQUENCE: 2

Met Ala Glu Glu. Asn His Thir Met Asn Glu Phe Ile Luell Thr Gly 1. 5 15

Phe Thir Asp His Pro Glu Lieu Lys Thir Luell Luell Phe Wall Wall Phe Phe 25 30

Ala Ile Tyr Lieu. Ile Thir Wal Wall Gly Asn Ile Ser Lell Wall Ala Lieu 35 4 O 45

Ile Phe Thir His Arg Arg Lieu. His Thir Pro Met Tyr Ile Phe Lieu. Gly SO 55 6 O

Asn Luell Ala Lieu Val Asp Ser Cys Ala Cys Ala Ile Thir Pro Llys 65 70 8O

Met Luell Glu Asn. Phe Phe Ser Glu Asn Lys Arg Ile Ser Luell Tyr Glu 85 90 95

Ala Wall Glin Phe Tyr Phe Leu Cys Thir Wall Glu Thir Ala Asp Cys 105 1 O

Phe Luell Luell Ala Ala Met Ala Tyr Asp Arg Wall Ala le Cys Asn 115 12 O 125

Pro Luell Glin Tyr His Ile Met Met Ser Lell le Gln Met 13 O 135 14 O

Thir Thir Gly Ala Phe Ile Ala Gly Asn Luell His Ser Met le His Wall 145 150 155 160

Gly Luell Wall Phe Arg Lieu Val Phe Gly Ser Asn His le Asn His 1.65 17O 17s

Phe Asp Ile Lieu Pro Lieu. Tyr Arg Luell Ser Asp Pro 18O 185 9 O

Ile Asn Glu Lieu Val Lieu. Phe Ile Phe Ser Gly Ser Wall Glin Wall 195 2O5

Phe Thir Ile Gly Ser Val Lieu. Ile Ser Luell Tyr Ile Luell Lieu. Thir 21 O 215 22O

Ile Phe Met Lys Ser Lys Glu Gly Arg Ala Ala Phe Ser Thr 225 23 O 235 24 O

Ala Ser His Phe Lieu. Ser Wall Ser Luell Phe Gly Ser Lieu. Phe 245 250 255 US 9,057,090 B2 15 16 - Continued

Phe Met Tyr Val Arg Pro Asn Lieu. Lieu. Glu Glu Gly Asp Lys Asp Ile 26 O 265 27 O

Pro Ala Ala Ile Leu Phe Thir Ile Wall Wall Pro Lieu. Lieu. ASn Pro Phe 27s 28O 285

Ile Tyr Ser Lieu. Arg Asn Arg Glu Wall Ile Ser Val Lieu. Arg Lys Ile 29 O 295 3 OO Lieu Met Lys Llys 3. OS

<210s, SEQ ID NO 3 &211s LENGTH: 681 &212s. TYPE: DNA <213> ORGANISM: Homosapiens 22 Os. FEATURE: 223 OTHER INFORMATION: RTP1S

<4 OOs, SEQUENCE: 3 atgtgcaagt ccctgacaac gggaga.gtgg aagaagatct tctacgagaa aatggaggag 6 O gtgaaacccg Cagact cotg ggacctgatc atggat.ccca acctic cago a taacg tattg 12 O gcc.ccc.ggat ggalagcagta Cctggagcag cacgcct citg gcc.gct tcca citgct cotgg 18O tgctgg cata gctggcagtic ctic ccaactg gtgat cotct tccacatgita cct ggataag 24 O acccagcgga C9ggctg.cgt. gcgcatgaga gtc.ttcaagc agctctgct a cagtgtggc 3OO t cct cocggc tiggacgagtic gtc catgctg gaggagalaca tagaggggct getggacaac 360 Ctcgtctgca gcctcc.ggga gcagtgctac ggggagaatg gggga cagta CC9catccac gtggCCtcCC gcc aagacCa C Cagcgc.cac C9gggagagt tctg.cgaggc Ctgcc.gc.ctg 480 ggcatcaccc actggaagcc cacggagaag atgctagagg aggaggcct C Cacct acacc 54 O ttct cocq go ctg.cgaatcc titccaagaca gcc.gact cq9 gttt cagctg tdactitctgc t ccctic cctt ggtgtatgtt Ctgggccacg gtgct Cttgc t catcatata cct gcagat c 660 t cct tcggca accctgtcta a. 681

SEQ ID NO 4 LENGTH: 226 TYPE PRT ORGANISM: Homo Sapiens FEATURE: OTHER INFORMATION: RTP1S

<4 OOs, SEQUENCE: 4 Met Cys Llys Ser Lieu. Thir Thr Gly Glu Trp Llys Ile Phe Tyr Glu 1. 5 1O 15

Lys Met Glu Glu Val Llys Pro Ala Asp Ser Trp Asp Lell Ile Met Asp 25 3O

Pro Asn Lieu Gln His Asn. Wall Lieu. Ala Pro Gly Trp Lys Gln Tyr Lieu. 35 4 O 45

Glu Glin His Ala Ser Gly Arg Phe His Cys Ser Trp Trp His Ser SO 55 6 O

Trp Glin Ser Ser Glin Leu Wall Ile Lieu. Phe His Met Lieu. Asp Llys 65 70 7s 8O

Thir Glin Arg Thr Gly Cys Val Arg Met Arg Val Phe Glin Lieu. Cys 85 90 95

Glu Cys Gly Ser Ser Arg Lieu. Asp Glu Ser Ser Met Lieu. Glu Glu 105 11 O

Asn Ile Glu Gly Lieu Val Asp Asn Lieu Val Cys Ser Lell Arg Glu Glin 115 12 O 125 US 9,057,090 B2 17 18 - Continued

Tyr Gly Glu Asn Gly Gly Glin Ile His Wall Ala Ser Arg 13 O 135 14 O

Glin Asp His Glin Arg His Arg Gly Glu Phe Cys Glu Ala Cys Arg Luell 145 150 155 160

Gly Ile Thr His Trp Lys Pro Thr Glu Lys Met Lieu. Glu Glu Glu Ala 1.65 17O 17s

Ser Thir Thir Phe Ser Arg Pro Ala Asn. Pro Ser Thir Ala Asp 18O 185 19 O

Ser Gly Phe Ser Asp Phe Cys Ser Leu Pro Trp. Cys Met Phe Trp 195 2O5

Ala Thr Wall Lieu. Luell Lell Ile Ile Luell Glin Ile Ser Phe Gly Asn 21 O 215

Pro Wall 225

2O The invention claimed is: ethylpyrazine, 2,5-dimethylpyrazine, 2,3-dimethylpyrazine, 1. A method to identify an agent for reducing an odor of a 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, pyrazine derivative, comprising: 2-monomethylpyrazine, 2-monoethylpyrazine, 2 ethyl-6- adding a test Substance and a pyrazine derivative repre methylpyrazine, 3-methyl-2-acetylpyrazine, and 3.5-dim sented by the following Formula (I): 25 ethyl-2-acetylpyrazine. 4. The method according to claim 1, wherein the olfactory (I) receptor OR5K1 is a protein consisting of an amino acid R4 N R sequence set forth in SEQID NO: 2. 30 5. The method according to claim 1, wherein the polypep 2 tide having at least 90% identity to the amino acid sequence of R N R SEQID NO: 2 is a polypeptide consisting of an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQID NO: 2 and having responsive wherein R represents methyl, ethyl, or acetyl; and R. R. and Reach independently represent hydrogen or methyl, to ness to the pyrazine derivative. a recombinant cell genetically engineered to express an olfac 6. The method according to claim 1, further comprising: tory receptor OR5K1 or a polypeptide which maintains measuring the response of the olfactory receptor or the responsiveness to a pyrazine derivative and which has at least polypeptide in the absence of the test Substance. 90% identity to the amino acid sequence of SEQID NO:2, in 7. The method according to claim 6, wherein, when the a cultured cell; 40 response of the olfactory receptor or the polypeptide in the measuring a response of the olfactory receptor to the pyra presence of a test substance is reduced to 60% or less of the Zine derivative; and response of the olfactory receptor or the polypeptide in the by comparing the response of the olfactory receptor or the absence of the test substance, the test substance is identified polypeptide in the presence of the test Substance to the as a Substance which inhibits response of the olfactory recep response of the olfactory receptor or the polypeptide to 45 tor or the polypeptide to the pyrazine derivative. the pyrazine derivative in the absence of the test sub 8. The method according to claim 1, wherein the step of stance, thereby identifying a test Substance inhibiting measuring a response of the olfactory receptor or the polypep the response of the olfactory receptor or the polypep tide is performed using measurement of intracellular cAMP tides on the basis of the measured response. level by ELISA or calcium imaging or reporter gene assay. 2. The method according to claim 1, wherein at least one of 9. The method according to claim 1, wherein the odor of the R to Ra represents an alkyl group. pyrazine derivative is an odor generated by reaction of a skin 3. The method according to claim 1, wherein the pyrazine tanning agent with skin. derivative is selected from the group consisting of 2,6-dim k k k k k