# Patent

## Title
Method of screening for an inhibitor of odor cause by furaneol

## Abstract

It is intended to identify a substance inhibiting an odor caused by 2,5-dimethyl-4-hydroxy-3(2H)-furanone. The present invention provides a method for searching for an inhibitor of an odor caused by 2,5-dimethyl-4-hydroxy-3(2H)-furanone, comprising: adding a test substance and 2,5-dimethyl-4-hydroxy-3(2H)-furanone to an olfactory receptor OR5K1 or a polypeptide having at least 80% amino acid sequence identity thereto; measuring the response of the olfactory receptor or the polypeptide to 2,5-dimethyl-4-hydroxy-3(2H)-furanone; and identifying a test substance inhibiting the response of the olfactory receptor or the polypeptide, on the basis of the measured response.

## Background
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The content of the electronically submitted substitute sequence listing, file name 2537—1030000_SequenceListing_ST25.txt, size 13,719 bytes; and date of creation Oct. 10, 2014, filed herewith, is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to a method for searching for an inhibitor of an odor caused by Furaneol.
BACKGROUND OF THE INVENTION
A large number of malodorous molecules differing in polarity or molecular weight are found in our living environments. Various deodorization methods have been developed so far in order to deodorize these diverse malodorous molecules. In general, such deodorization methods are broadly classified into biological methods, chemical methods, physical methods, and sensory methods. Among the malodorous molecules, highly polar short-chain fatty acids or amines can be deodorized by the chemical method, i.e., neutralization reaction. Sulfur compounds such as thiol can be deodorized by the physical method, i.e., adsorption treatment. Still, there remain a large number of malodorous molecules which cannot be dealt with by the conventional deodorization methods. Also, the deodorization method based on adsorption treatment has a problem that it tends to cause the re-emission of a bad smell. In addition, these conventional methods may mask even odors other than the bad smell of interest. Thus, a deodorization method which can overcome these problems has been demanded.
Another known method involves deodorizing a bad smell by rendering a different odor more strongly perceivable using a fragrance. In this method, however, the odor of the fragrance may cause discomfort. In addition, an odorous substance which exhibits an effective deodorizing effect on the malodorous substance of interest must be searched for in order to mask the bad smell by a different odor of a perfume, a fragrance, or the like. Heretofore, sensory tests by experts have been conducted for the evaluation of odors. Such sensory tests, however, has problems such as the need of fostering experts capable of evaluating odors and low throughputs. Thus, the previous search for an odorous substance which exhibits a deodorizing effect has not been easy to achieve.
In mammals such as humans, the sense of smell works by the mechanism where odor molecules bind to olfactory receptors on olfactory nerve cells present in the olfactory epithelium located in the upper region of the nasal cavity to transmit the responses of the receptors thereto to the central nervous system. In humans, approximately 400 olfactory receptors have been reported, and genes encoding these receptors account for approximately 3% of all human genes. In general, a set of olfactory receptors is associated with a set of odor molecules. This means that individual olfactory receptors can respond to a plurality of structurally similar odor molecules with different affinities, while individual odor molecules can be recognized by a plurality of olfactory receptors. According to another report, an odor molecule activating a certain olfactory receptor functions as an antagonist inhibiting the activation of a different olfactory receptor. These combined responses of a set of olfactory receptors render individual odors perceivable.
Accordingly, when a certain odor molecule coexists with a different odor molecule, the different odor molecule may inhibit the response of a receptor corresponding to the certain odor molecule, resulting in the final perception of a different odor. Such a mechanism is called the antagonism of an olfactory receptor. The alteration of an odor caused by this receptor antagonism is a preferred deodorization approach because this approach, unlike the deodorization methods which involve adding another odor of, for example, a perfume, and fragrance can specifically cancel the perception of a bad smell and does not cause discomfort derived from such a fragrance.
Skin tanning agents (also called self-tanning agents or sunless tanning agents) are skin cosmetics which colors the skin. Mainly, dihydroxyacetone (DHA) is used alone or in combination with erythrulose or the like as an ingredient which causes the skin color to turn brown. Such an ingredient reacts with the upper layer of the skin to color the skin brown. Although this coloring is believed to proceed through browning reaction, the details of a mechanism underlying this reaction have hardly been elucidated. The browning reaction is also called Maillard reaction in the field of food chemistry. This term refers to the reaction through which a nitrogen-containing compound such as an amino acid or a protein is polymerized with a reduced sugar to form a brown polymer called melanoidin. The Maillard reaction is involved in food coloring or aroma component formation caused by the heating, etc. of foods.
2,5-Dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) is a substance known to have an odor expressed as a “strong fruity caramel aroma”, a “burnt caramel aroma”, “burnt sugar”, a “curry-like flavor”, a “cotton candy smell”, etc. (Non-Patent Documents 1 to 4). Patent Document 1 describes a beer-tasted beverage having a taste and flavor or a fragrant smell enhanced by the formation of maltol and Furaneol in a fermentation undiluted solution.
CITATION LIST
Patent Document


    [Patent Document 1] WO 2009/078360


Non-Patent Document


    [Non-Patent Document 1] Gosei Koryo—Kagaku To Shohin Chishiki—(Synthetic Flavor and Fragrance—Chemistry and Product Knowledge—in English), 2005, The Chemical Daily Co., Ltd.
    [Non-Patent Document 2] J. Agric. Food Chem., 1997, 45 (6): 2217-2224
    [Non-Patent Document 3] ACS Symp Ser., 2002, 836: 108-123
    [Non Patent Document 4] Anal Chim Acta., 2010, 657 (2): 198-203


SUMMARY OF INVENTION
The present invention provides:
a method for searching for an inhibitor of an odor caused by 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol), comprising:
adding a test substance and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) to an olfactory receptor OR5K1 or a polypeptide having at least 80% amino acid sequence identity thereto;
measuring the response of the olfactory receptor or the polypeptide to 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol); and
identifying a test substance inhibiting the response of the olfactory receptor or the polypeptide, on the basis of the measured response.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the responses of olfactory receptors to Furaneol. The abscissa represents individual olfactory receptors. The ordinate represents response intensity.
FIG. 2 shows the response of an olfactory receptor OR5K1 to varying concentrations of Furaneol. n=3, error bar=±SE.
FIG. 3 shows results of sensory evaluation on the inhibitory effects of various compounds on the odor of Furaneol. n=3, error bar=±SE.



DETAILED DESCRIPTION OF THE INVENTION
The term “masking” in relation to odors as used herein refers to a general approach for canceling or weakening the perception of the odor of interest. The “masking” may include chemical approaches, physical approaches, biological approaches, and sensory approaches. Examples of the masking include: an arbitrary approach for removing an odor molecule causative of the odor of interest from the environment (e.g., adsorption and chemical decomposition of the odor molecule); an approach for preventing the odor of interest from being released to the environment (e.g., containment); and a method which involves adding a different odor of a flavor, a fragrance, or the like to reduce the perception of odor of interest.
The term “masking based on olfactory receptor antagonism” as used herein refers to one form of the above-mentioned “masking” in a broad sense. This approach utilizes both an odor molecule of the odor of interest and a different odor molecule to thereby inhibit a receptor response to the odor molecule of interest by the different odor molecule, resulting in change in odor which is perceived by individuals. The masking based on olfactory receptor antagonism is distinct from the approach of canceling the odor of interest by a different strong odor of a fragrance or the like, even though these approaches both employ the different odor molecule. One example of the masking based on olfactory receptor antagonism is the case of using a substance, such as an antagonist, which inhibits the response of an olfactory receptor. A substance inhibiting the response of a receptor to an odor molecule responsible for a particular odor can be applied to the receptor to thereby inhibit the response of the receptor to the odor molecule. The odor which is finally perceived by individuals can therefore be changed.
The term “Furaneol” as used herein refers to 2,5-dimethyl-4-hydroxy-3(2H)-furanone. The term “odor caused by Furaneol” can be an odor brought about by 2,5-dimethyl-4-hydroxy-3(2H)-furanone. The “odor caused by Furaneol” or the “odor caused by 2,5-dimethyl-4-hydroxy-3(2H)-furanone” as used herein can be typically expressed as, for example, a caramel aroma or a burnt sugar smell. Also, the “odor caused by Furaneol” or the “odor caused by 2,5-dimethyl-4-hydroxy-3(2H)-furanone” as used herein can be an unpleasant smell generated by the application of a conventional self-tanning agent to the skin, more specifically, an unpleasant smell expressed as a “burnt sugar smell” or the like generated by the application of a skin tanning agent containing dihydroxyacetone (DHA) to the skin.
The reported problem of commercially available skin tanning agents (self-tanning agents or sunless tanning agents) is their unique unpleasant smells expressed as earthy, burnt sugar smells, etc. upon application (D. M. Hindenlang and M. E. McDonnell, Cosmetics & Toiletries magazine, 2008, Vol. 123, No. 7, p. 67-74). Thus, improvement in the smells has been demanded. As a result of examining a cause of the unpleasant smell, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) produced via methylglyoxal from dihydroxyacetone (DHA) contained in the skin tanning agents has been found to be a causative substance. For reducing such an unpleasant smell attributed to Furaneol, it has been required to control the odor of Furaneol.
The present inventors searched for an olfactory receptor which responds to Furaneol and successfully identified the receptor. The present inventor has also found that a substance inhibiting the response of the olfactory receptor can inhibit the perception of an odor caused by Furaneol by masking based on olfactory receptor antagonism. On the basis of these findings, the present inventor has found that a substance controlling an odor caused by Furaneol can be searched for with the response of the olfactory receptor as an index.
In the present specification, the sequence identity between nucleotide sequences or amino acid sequences is calculated according to the Lipman-Pearson method (Science, 1985, 227: 1435-41). Specifically, the sequence identity is calculated by analysis using the search homology program (Unit size to compare (ktup): 2) of genetic information processing software Genetyx-Win (Ver. 5.1.1; Software Development Co., Ltd.).
As shown in FIG. 1, the present inventor identified an olfactory receptor OR5K1 as only one receptor responsive to Furaneol from among many olfactory receptors. OR5K1 is a novel receptor for Furaneol which has not been found so far to respond to Furaneol. As shown in FIG. 2, OR5K1 responds to Furaneol in a concentration-dependent manner. Thus, a substance inhibiting the response of OR5K1 can change the perception of an odor caused by Furaneol in the central nervous system by masking based on olfactory receptor antagonism, resulting in the selective inhibition of the odor caused by Furaneol.
According to the present invention, an inhibitor of an odor caused by Furaneol can be efficiently searched for. The inhibitor of an odor caused by Furaneol, identified by the present invention, can selectively deodorize the odor caused by Furaneol by masking based on olfactory receptor antagonism. Thus, the inhibitor of an odor caused by Furaneol, identified by the present invention can deodorize the odor caused by Furaneol, for example, an unpleasant smell generated upon application of conventional self-tanning agents (also called sunless tanning agents), without causing the problems such as discomfort derived from the odor of a fragrance in conventional deodorization methods using a deodorizer or a fragrance.
Thus, the present invention provides a method for searching for an inhibitor of an odor caused by Furaneol. This method comprises: adding a test substance and Furaneol to an olfactory receptor OR5K1; measuring the response of the olfactory receptor to Furaneol; and identifying a test substance inhibiting the response of the olfactory receptor, on the basis of the measured response. The identified test substance is selected as an inhibitor of an odor caused by Furaneol. The method of the present invention can be a method performed in vitro or ex vivo.
In the method of the present invention, a test substance and the odor causative substance Furaneol are added to an olfactory receptor OR5K1. A commercially available product (e.g., FURANEOL®; Nihon Firmenich K.K.) can be purchased and used as Furaneol.
The test substance used in the method of the present invention is not particularly limited as long as the substance is desired to be used as an inhibitor of an odor caused by Furaneol. The test substance may be a naturally occurring substance or may be a substance artificially synthesized by a chemical or biological method or the like. Alternatively, the test substance may be a compound, a composition, or a mixture.
The olfactory receptor OR5K1 used in the method of the present invention refers to an olfactory receptor expressed on a human olfactory cell and is registered as GI: 115270955 in GenBank. OR5K1 is encoded by a gene having the nucleotide sequence represented by SEQ ID NO: 1. This protein consists of the amino acid sequence represented by SEQ ID NO: 2.
In the method of the present invention, the olfactory receptor OR5K1 can be used in an arbitrary form unless the form loses responsiveness to Furaneol. For example, the olfactory receptor can be used in a form including: tissues or cells naturally expressing the olfactory receptor, such as an osmoreceptor or olfactory cells isolated from an organism, or cultures thereof; the membranes of olfactory cells carrying the olfactory receptor; recombinant cells genetically engineered to express the olfactory receptor, or cultures thereof; the membranes of the recombinant cells having the olfactory receptor; and artificial lipid bilayers having the olfactory receptor. These forms are all included in the scope of the olfactory receptor used in the present invention.
According to a preferred aspect, a cell naturally expressing the olfactory receptor, such as an olfactory cell, or a recombinant cell genetically engineered to express the olfactory receptor, or cultures of any of these cells are used as the olfactory receptor OR5K1. The recombinant cell can be prepared by the transformation of a cell with a vector having an insert of the gene encoding the olfactory receptor.
Preferably, a receptor-transporting protein (RTP) gene, together with the gene of the olfactory receptor, is transferred to the cell in order to promote the expression of the olfactory receptor on the cell membrane. Preferably, an RTP1S gene, more preferably RTP1S and RTP2 genes, together with the gene of the olfactory receptor, are transferred to the cell. Examples of RTP1S and RTP2 include human RTP1S and human RTP2, respectively. The human RTP1S is registered as GI: 50234917 in GenBank. This protein is encoded by a gene having the gene sequence represented by SEQ ID NO: 3 and consists of the amino acid sequence represented by SEQ ID NO: 4. The human RTP2 is registered as GI: 258547120 in GenBank. This protein is encoded by a gene having the gene sequence represented by SEQ ID NO: 5 and consists of the amino acid sequence represented by SEQ ID NO: 6.
Alternatively, a polypeptide which consists of an amino acid sequence having at least 78%, for example, 80% or higher, preferably 85% or higher, more preferably 90% or higher, still preferably 95% or higher, even more preferably 98% or higher, still even more preferably 99% or higher sequence identity to the amino acid sequence (SEQ ID NO: 4) of human RTP1S or the amino acid sequence (SEQ ID NO: 6) of human RTP2 and promotes the expression of the olfactory receptor on the cell membrane, as with human RTP1S or RTP2, may be used instead of the human RTP1S or RTP2. For example, a human RTP1S variant which is encoded by a gene having the gene sequence represented by SEQ ID NO: 7 and consists of the amino acid sequence represented by SEQ ID NO: 8 has 78.9% sequence identity to the amino acid sequence of human RTP1S represented by SEQ ID NO: 4 and has the function of promoting the expression of the olfactory receptor on the cell membrane. Alternatively, mouse RTP1S (Sci Signal., 2009, 2: ra9) also has 89% sequence identity to the amino acid sequence of human RTP1S represented by SEQ ID NO: 4 and has the function of promoting the expression of the olfactory receptor on the cell membrane. Such a human RTP1S variant and mouse RTP1S can be used instead of the human RTP1S in the preparation of the recombinant cell expressing the olfactory receptor as mentioned above. Alternatively, an RTP1S variant polypeptide which has at least 80%, for example, 80% or higher, preferably 85% or higher, more preferably 90% or higher, still preferably 95% or higher, even more preferably 98% or higher, still even more preferably 99% or higher amino acid sequence identity to the human RTP1S variant consisting of the amino acid sequence represented by SEQ ID NO: 8 or the mouse RTP1S and promotes the expression of the olfactory receptor on the cell membrane may also be used instead of the human RTP1S in the preparation of the recombinant cell expressing the olfactory receptor as mentioned above.
According to the method of the present invention, the response of the olfactory receptor OR5K1 to Furaneol is measured following the addition of the test substance and Furaneol to the olfactory receptor. This measurement can be performed by an arbitrary method known in the art as a method for measuring the response of the olfactory receptor, for example, the measurement of an intracellular cAMP level. For example, the olfactory receptor, when activated by an odor molecule, is known to be conjugated with intracellular Gas to activate adenylate cyclase, thereby increasing intracellular cAMP levels (Mombaerts P. Nat Neurosci. 5, 263-278). Thus, the intracellular cAMP level after the addition of the odor molecule can be used as an index for measuring the response of the olfactory receptor. Examples of methods for measuring the cAMP level include ELISA and reporter gene assay. Another example of the method for measuring the response of the olfactory receptor includes calcium imaging.
Subsequently, the effect of the test substance on the response to Furaneol is evaluated on the basis of the measured response of the olfactory receptor to identify a test substance inhibiting the response. The evaluation on the effect of the test substance can be conducted, for example, by comparing the responses of the receptor to Furaneol measured in the presence of various concentrations of the test substance. As a more specific example, the responses of the receptor to Furaneol are compared between a group supplemented with a higher concentration of the test substance and a group supplemented with a lower concentration of the test substance, between a group supplemented with the test substance and a group non-supplemented with the test substance, between a group supplemented with the test substance and a group supplemented with a control substance, or between before and after the addition of the test substance. When the addition of the test substance or the addition of a higher concentration of the test substance inhibits the response of the receptor, this test substance can be identified as a substance inhibiting the response of the olfactory receptor to Furaneol.
In the method of the present invention, a polypeptide having a function equivalent to OR5K1 can be used as the olfactory receptor instead of OR5K1. Examples of the polypeptide include a polypeptide which consists of an amino acid sequence having at least 80%, for example, 80% or higher, preferably 85% or higher, more preferably 90% or higher, still preferably 95% or higher, even more preferably 98% or higher, still even more preferably 99% or higher sequence identity to the amino acid sequence (SEQ ID NO: 2) of OR5K1 and is responsive to Furaneol.
Alternatively, in the method of the present invention, OR5K1 mentioned above as the olfactory receptor and polypeptides having a function equivalent thereto may each be used alone or may be used in combination of any two or more thereof.
The test substance identified by the above procedures is a substance which can inhibit the individual's perception of an odor caused by Furaneol by inhibiting the response of the olfactory receptor to Furaneol. Thus, the test substance identified by the above procedures is selected as an inhibitor of an odor caused by Furaneol. For example, when the receptor response in a test substance-supplemented group measured by the above procedures is reduced to preferably 60% or less, more preferably 50% or less, further preferably 25% or less of that in a test substance-non-supplemented group (e.g., the above-mentioned group non-supplemented with the test substance, group supplemented with a control substance, or before the addition of the test substance), the test substance can be selected as an inhibitor of an odor caused by Furaneol.
The substance selected by the method of the present invention can inhibit the odor caused by Furaneol, by olfactory masking based on the inhibition of the response of the olfactory receptor to Furaneol.
Thus, in one embodiment, the substance selected by the method of the present invention can serve as an active ingredient in an inhibitor of an odor caused by Furaneol. Alternatively, the substance selected by the method of the present invention can be contained as an active ingredient for inhibiting an odor caused by Furaneol in a compound or a composition for inhibiting an odor caused by Furaneol. Alternatively, the substance selected by the method of the present invention can be used for production of an inhibitor of an odor caused by Furaneol or for production of a compound or a composition for inhibiting an odor caused by Furaneol.
In one embodiment, the substance selected by the method of the present invention can be used as an active ingredient for inhibiting an odor caused by Furaneol, for example, a caramel aroma, a burnt sugar smell, or an unpleasant smell (e.g., a burnt sugar smell) generated by the application of a self-tanning agent to the skin or upon application of a product containing DHA.
In one embodiment, the substance selected by the method of the present invention can be used as an active ingredient for inhibiting an odor caused by Furaneol in every compound or composition desired to inhibit an odor caused by Furaneol or in every environment desired to inhibit an odor caused by Furaneol. Alternatively, the substance selected by the method of the present invention can be used as an active ingredient for inhibiting an odor caused by Furaneol, for production of a compound or a composition desired to inhibit an odor caused by Furaneol. Examples of the compound or the composition desired to inhibit an odor caused by Furaneol include skin tanning agents (also called self-tanning agents or sunless tanning agents), for example, a skin tanning agent containing DHA as a coloring agent and other skin tanning agents which employ browning reaction, and other products containing DHA. Furaneol excessively contained in a food or a drink may work as an off-flavor. For example, the presence of excessive Furaneol in powdered milk reduces its flavor or taste. Thus, other examples of the compound or the composition desired to inhibit an odor caused by Furaneol include foods or drinks desired to reduce the odor of Furaneol and compositions containing the foods or the drinks.
The following composition, production method, use, or method will be further disclosed herein as an exemplary embodiment of the present invention. However, the present invention is not intended to be limited by these embodiments.
<1> A method for searching for an inhibitor of an odor caused by Furaneol, comprising:
adding a test substance and Furaneol to an olfactory receptor OR5K1 or a polypeptide having at least 80% amino acid sequence identity thereto;
measuring the response of the olfactory receptor or the polypeptide to Furaneol; and
identifying a test substance inhibiting the response of the olfactory receptor or the polypeptide, on the basis of the measured response.
<2> The method according to <1>, wherein the olfactory receptor OR5K1 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
<3> The method according to <1> or <2>, wherein the polypeptide having at least 80% amino acid sequence identity to the olfactory receptor OR5K1 is a polypeptide having preferably at least 85%, more preferably at least 90%, still preferably at least 95%, even more preferably at least 98%, still even more preferably at least 99% amino acid sequence identity to the olfactory receptor OR5K1.
<4> The method according to any one of <1> to <3>, wherein the polypeptide having at least 80% amino acid sequence identity to the olfactory receptor OR5K1 is a polypeptide which has preferably at least 85%, more preferably at least 90%, still preferably at least 95%, even more preferably at least 98%, still even more preferably at least 99% amino acid sequence identity to the olfactory receptor OR5K1 and is responsive to Furaneol.
<5> The method according to any one of <1> to <4>, wherein, preferably, the olfactory receptor OR5K1 or the polypeptide having at least 80% amino acid sequence identity thereto is expressed on a recombinant cell genetically engineered to express the olfactory receptor or the polypeptide.
<6> The method according to <5>, wherein, preferably, the recombinant cell is the following cell:
a cell cotransfected with a gene of the olfactory receptor or the polypeptide and an RTP1S gene;
a cell cotransfected with a gene of the olfactory receptor or the polypeptide and RTP1S and RTP2 genes;
a cell cotransfected with a gene of the olfactory receptor or the polypeptide and a gene encoding a polypeptide which consists of an amino acid sequence having at least 78%, preferably 80% or higher, more preferably 85% or higher, still preferably 90% or higher, even more preferably 95% or higher, still even more preferably 98% or higher, further preferably 99% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 4 and promotes the expression of the olfactory receptor on the membrane, as with human RTP1S; or
a cell cotransfected with a gene of the olfactory receptor or the polypeptide and a gene encoding a human RTP1S variant.
<7> The method according to <5> or <6>, wherein, preferably, cultures of the recombinant cell are used as the olfactory receptor OR5K1 or the polypeptide having at least 80% amino acid sequence identity thereto.
<8> The method according to any one of <1> to <7>, preferably further comprising measuring the response of the olfactory receptor or the polypeptide in the absence of the test substance.
<9> The method according to <8>, preferably further comprising the following:
when the response of the olfactory receptor or the polypeptide in the presence of the test substance is reduced compared with the response of the olfactory receptor or the polypeptide in the absence of the test substance, the test substance is identified as a substance inhibiting the response of the receptor or the polypeptide to Furaneol.
<10> The method according to <8>, preferably further comprising the following:
when the response of the olfactory receptor or the polypeptide in the presence of the test substance is reduced to preferably 60% or less, more preferably 50% or less, further preferably 25% or less, of the response of the olfactory receptor or the polypeptide in the absence of the test substance, the test substance is identified as a substance inhibiting the response of the olfactory receptor or the polypeptide to Furaneol.
<11> The method according to any one of <1> to <10>, wherein the step of measuring the response of the olfactory receptor or the polypeptide is the measurement of an intracellular cAMP level by ELISA or reporter gene assay, or calcium imaging.
EXAMPLES
Hereinafter, the present invention will be described more specifically with reference to Examples.
Example 1
Identification of Olfactory Receptor Responding to Furaneol
1) Cloning of Human Olfactory Receptor Gene
On the basis of sequence information registered in GenBank, each human olfactory receptor gene was cloned by PCR with Human Genomic DNA Female (G1521: Promega Corp.) as a template. Each gene thus amplified by PCR was inserted into a pENTR vector (Invitrogen Corp.) according to the manual. A NotI-AscI site present on the pENTR vector was recombined into a NotI-AScI site prepared downstream of a Flag-Rho tag sequence on a pME18S vector.
2) Preparation of pME18S-Human RTP1S Vector
A human RTP1S gene (SEQ ID NO: 3) encoding human RTP1S (SEQ ID NO: 4) was inserted into the EcoRI-XhoI site of the pME18S vector.
3) Preparation of Olfactory Receptor-Expressing Cell
HEK293 cells expressing each of 400 types of human olfactory receptors were prepared. Each reaction solution having the composition shown in Table 1 was prepared, then left standing for 15 minutes in a clean bench, and then added to each well of a 96-well plate (Becton, Dickinson and Company). Subsequently, the HEK293 cells (3×105 cells/cm2) were inoculated thereto at 100 μL/well and then cultured at 37° C. for 24 hours in an incubator kept at 5% CO2.











TABLE 1









OPTI-MEM (GIBCO)
50 
μL



Human olfactory receptor gene
0.075
μg



(inserted to N-terminally Flag-



Rho-tagged pME18S vector)



pGL4.29 (fluc2P-CRE-hygro,
0.03
μg



Promega)



pGL4.75 (hRluc-CMV, Promega)
0.03
μg



pME18S-human RTP1S vector
0.03
μg



lipofectamine 2000 (Invitrogen)
0.4
μL










4) Luciferase Assay

The olfactory receptors expressed on the HEK293 cells are conjugated with intracellular Gαs to activate adenylate cyclase, thereby increasing intracellular cAMP levels. In this study, their responses to Furaneol were measured using luciferase reporter gene assay which involved monitoring increase in intracellular cAMP level as luminescence intensity derived from a firefly luciferase gene (fluc2P-CRE-hygro). In addition, the cells were cotransfected with the reporter gene and a fusion gene (hRluc-CMV) of a Renilla luciferase gene downstream of a CMV promoter for use as an internal standard for correcting transfection efficiency or an error in the number of cells.
A medium was removed from the cultures prepared in the preceding paragraph 3). To the resulting cultures, 75 μL of a solution containing Furaneol (3 mM) prepared with a CD293 medium (Invitrogen Corp.) was added. The cells were cultured for 2.5 hours in a CO2 incubator to express the luciferase genes at sufficient levels in the cells. The luciferase activity was measured using Dual-GloTM luciferase assay system (Promega Corp.) according to the instruction manual of the product. Firefly luciferase-derived luminescence intensity induced by stimulation with Furaneol was divided by luminescence intensity in the cells without Furaneol stimulation to calculate a value as fold increase, which was in turn used as an index for response intensity.
5) Results
As a result of measuring the responses of 400 types of olfactory receptors to Furaneol (3 mM), only the olfactory receptor OR5K1 exhibited a response to Furaneol (FIG. 1). OR5K1 is a novel Furaneol receptor which has not been found so far to respond to Furaneol.
Example 2
Concentration-Dependent Response of OR5K1 to Furaneol
The olfactory receptor OR5K1 (SEQ ID NO: 2) was expressed, together with human RTP1S (SEQ ID NO: 4), on HEK293 cells by the same procedures as in Example 1 and then examined for the concentration dependence of its response to varying concentrations of Furaneol (0, 3, 10, 30, 100, 300, 1000, and 3000 μM). As a result, OR5K1 exhibited a concentration-dependent response to Furaneol (FIG. 2).
Example 3
Identification of OR5K1 Antagonist
84 types of test substances were examined for their antagonistic activity against the response of the olfactory receptor OR5K1 to Furaneol.
Furaneol (3 mM) and each test substance (100 μM) were added to HEK293 cells allowed to express the olfactory receptor OR5K1 by the same procedures as in Example 2. The response of the olfactory receptor was measured to evaluate change in receptor response caused by the addition of the test substance.
The rate of inhibition of the receptor response by the test substance was calculated as follows: firefly luciferase-derived luminescence intensity (Y) in the olfactory receptor OR5K1-expressing cells without Furaneol stimulation was subtracted from firefly luciferase-derived luminescence intensity (X) induced by stimulation with Furaneol alone to determine receptor activity (X−Y) based on the stimulation with Furaneol alone. Likewise, the luminescence intensity (Y) in the cells without Furaneol stimulation was subtracted from luminescence intensity (Z) induced by stimulation with the mixture of Furaneol and the test substance to determine receptor activity (Z−Y) in the presence of the test substance. The rate of reduction in the receptor activity (Z−Y) in the presence of the test substance with respect to the receptor activity (X−Y) based on the stimulation with Furaneol alone was calculated according to an expression shown below to determine the rate of inhibition of the receptor response by the test substance. In this assay, a plurality of independent duplicate experiments were performed. An average of the experiments in each run was obtained.

Rate of inhibition (%)={1−(Z−Y)/(X−Y)}×100

As a result, 17 types of test substances had 40% or higher rate of inhibition of the response of OR5K1 to Furaneol (reduced the response to 60% or less), demonstrating that these test substances have antagonistic activity against OR5K1 (Table 2).










TABLE 2









75% or more rate of
ω-6-Hexadecenlactone (ambrettolide)



inhibition (response
7-Acetyl-1,2,3,4,5,6,7,8-octahydro-1,1,6,7-



was reduced to 25%
tetramethyl-naphthalene (Iso E Super)



or less; very strong
3-(4-tert-Butylphenyl)propanal (bourgeonal)



antagonist)
5-Methyl-2-(1-methylethyl)-phenol (thymol)




Oxacyclohexadecen-2-one (Habanolide(R))




(5E)-3-Methylcyclopentadec-5-en-1-one




(Muscenone(R) Delta)




Citral




Patchouli oil




1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-




hexamethyl-2-naphthalenyl)-ethanone




(Tonalid(R))




4,6,6,7,8,8-Hexamethyl-1,3,4,6,7,8-




hexahydrocyclopenta[g]isochromene




(galaxolide)




(E)-3-Methyl-4-(2,6,6-trimethylcyclohex-2-




en-1-yl)but-3-en-2-one (methyl ionone-G)




Muscone




1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-




tetramethyl-1H-3a,7-methanoazulen-5-yl)-




ethanone (acetylcedrene)



50% or more rate of
Cedryl acetate



inhibition
2-Ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-



(response was
yl)-2-buten-1-ol (bangalol)



reduced to 50% or



less; strong



antagonist)



40% or more rate of
Lemon oil



inhibition
(1-Methyl-2-(1,2,2-trimethylbicyclo[3.1.0]-



(response was
hex-3-ylmethyl)cyclopropyl)methanol



reduced to 60% or
(Javanol(R))



less; weak



antagonist)










Example 4
Evaluation on Ability of OR5K1 Antagonist to Inhibit Odor of Furaneol
Each test substance having antagonistic activity against OR5K1, identified in Example 3, was confirmed by a sensory test for its ability to inhibit the odor of Furaneol.
Each panelist smelled 0.5 g of Furaneol (1%)-containing cloth supplemented with 0.5 μL of a flavor, and then evaluated the intensity of the odor of Furaneol compared with cloth non-supplemented with the flavor. The sensory evaluation test was conducted by 3 panelists. The odor of Furaneol was evaluated as 1 when strongly perceivable and as 5 when not perceivable.
As a result, all the 17 types of test substances demonstrated to inhibit the response of OR5K1 to Furaneol in Example 3 inhibited the odor of Furaneol (FIG. 3).

## Claims

1. A method of screening for an inhibitor of an odor caused by 2,5-dimethyl-4-hydroxy-3(2H)-furanone, comprising:
(1), adding a test substance and 2,5-dimethyl-4-hydroxy-3(2H)-furanone to a cultured cell expressing olfactory receptor OR5K1, wherein the amino acid sequence of olfactory receptor OR5K1 consists of the amino acid sequence of SEQ ID NO:2, or a polypeptide having at least 95% amino acid sequence identity thereto;
(2) measuring the response of the olfactory receptor or the polypeptide to 2,5-dimethyl-4-hydroxy-3(2H)-furanone; and
(3) identifying a test substance that inhibits the response measured in (2) as an inhibitor of the odor.

2. The method according to claim 1, wherein the cultured cell expresses olfactory receptor OR5K1 that has an amino acid sequence that consists of the amino acid sequence of SEQ ID NO: 2.

3. The method according to claim 1, wherein the polypeptide having at least 95% amino acid sequence identity to the olfactory receptor OR5K1 is a polypeptide which consists of an amino acid sequence having at least 95% identity to the amino acid sequence represented by SEQ ID NO: 2 and is responsive to 2,5-dimethyl-4-hydroxy-3(2H)-furanone.

4. The method according to claim 1, wherein the olfactory receptor OR5K1 or the polypeptide having at least 95% amino acid sequence identity thereto is expressed on a recombinant cell genetically engineered to express the olfactory receptor or the polypeptide.

5. The method according to claim 1, further comprising measuring the response of the olfactory receptor or the polypeptide in the absence of the test substance.

6. The method according to claim 5, wherein when the response of the olfactory receptor or the polypeptide in the presence of the test substance is reduced to 60% or less of the response of the olfactory receptor or the polypeptide in the absence of the test substance, the test substance is identified as a substance inhibiting the response of the olfactory receptor or the polypeptide to 2,5-dimethyl-4-hydroxy-3(2H)-furanone.

7. The method according to claim 1, wherein the step of measuring the response of the olfactory receptor or the polypeptide is the measurement of an intracellular cAMP level by ELISA or reporter gene assay, or calcium imaging.

8. The method according to claim 1, wherein the cultured cell expressing the olfactory receptor OR5K1 or the polypeptide having at least 95% amino acid sequence identity thereto is a recombinant cell genetically engineered to express the olfactory receptor or the polypeptide.

9. The method according to claim 1, wherein the cultured cell expresses a polypeptide having at least 95% amino acid sequence identity to the olfactory receptor OR5K1.

10. The method according to claim 9, wherein the polypeptide has at least 98% amino acid sequence identity to olfactory receptor OR5K1.

11. The method according to claim 10, wherein the polypeptide has at least 99% amino acid sequence identity to olfactory receptor OR5K1.