JP7214486B2 - Search method for cat urine odor inhibitor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a search method for cat urine odor inhibitors.

2. Description of the Related Art In a disposal tool for treating pet excrement, a deodorant is placed in the disposal tool for the purpose of making the odor generated from the excrement imperceptible. For example, Patent Document 1 discloses a pet deodorant containing an aluminosilicate represented by a specific chemical formula. Patent Document 2 also discloses a deodorant-containing product containing a deodorant containing a phenolic compound and/or mediator and an enzyme capable of oxidizing them. Patent Document 3 discloses that a β-glucuronidase inhibitor containing a macrocyclic ketone as an active ingredient suppresses the production of volatile urinary odor components caused by the action of β-glucuronidase on urine, thereby reducing the urinary odor of humans and animals. It is stated that the suppression of

Cat urine odor (hereinafter also referred to as cat urine odor) is known to be more odorous than human and dog urine odors, and an improvement in the deodorizing effect against cat urine odor is desired. However, it is difficult to say that the pet deodorants, deodorant-containing products, and β-glucuronidase inhibitors described in Patent Documents 1 to 3 have a sufficient deodorizing effect on cat urine odor. The foul-smelling components in cat urine include thiols specific to cat urine, such as 3-mercapto-3-methylbutylformate (MMF) and 3-mercapto-3-methyl-1-butanol (MMB). (Non-Patent Documents 1 and 2). Therefore, in order to suppress cat urine odor, suppression of the odor of these thiol compounds is required.

In mammals such as humans, odor molecules bind to olfactory receptors on olfactory nerve cells present in the olfactory epithelium of the upper nasal cavity, and the responses of these receptors are transmitted to the central nervous system. recognized by It has been reported that there are about 400 olfactory receptors in humans, and the genes encoding them account for about 2% of all human genes. Generally, olfactory receptors and odor molecules are associated in many-to-many combinations. That is, each olfactory receptor receives multiple structurally similar odorants with different affinities, while each odorant molecule is received by multiple olfactory receptors. Furthermore, it has also been reported that an odorant molecule that activates one olfactory receptor acts as an antagonist that inhibits the activation of another olfactory receptor. The combination of responses from these multiple olfactory receptors results in the perception of individual odors.

Therefore, even when the same odorant molecule is present, if another odorant molecule is present at the same time, the receptor response is inhibited by the other odorant molecule, and the final perceived odor may be completely different. This mechanism is called olfactory receptor antagonism. Suppression of odors by this receptor antagonism is different from deodorization by adding other odors such as perfumes and air fresheners. It has the advantage of not causing discomfort due to the smell of

Based on the concept of olfactory receptor antagonism, several methods have been disclosed so far for identifying malodor suppressing substances using olfactory receptor activity as an index. For example, Patent Document 4 discloses a search for substances that suppress the malodorous odors of hexanoic acid and isovaleric acid, using the activity of olfactory receptors that specifically respond to these malodorous substances as an index. Similarly, Patent Document 5 describes a substance that suppresses the odor of skatole, and Patent Document 6 describes a substance that suppresses the smell of polysulfide compounds. It is disclosed to search as Patent Document 7 discloses that olfactory receptors OR1A1, OR2W1, OR4S2, OR5K1, OR5P3, OR8H1, OR9Q2, OR10G4 and OR10G7 respond to urine odor-causing substances such as p-cresol and 2-methoxy-4-vinylphenol. is described. Non-Patent Document 3 states that the olfactory receptor OR2M3 specifically responds to 3-mercapto-2-methylpentan-1-ol, an odorant contained in onions, and that this receptor is weaker in intensity. Other thiols such as 3-mercapto-3-methylhexan-1-ol and similar It is stated that it did not respond to the substance. However, there have been no reports of techniques for suppressing cat urine-derived odors, particularly thiol odors, based on olfactory receptor antagonism.

JP 2007-229151 A JP-A-2005-65750 JP 2014-195696 A JP 2012-050411 A JP 2012-249614 A International Publication No. 2016/204211 JP 2015-211667 A

Chem Biol, 2006, 13(10): 1071-1079 J Chem Ecol, 2018, 44(4): 364-373 Chemical Senses, 2017, 42(3): 195-210

INDUSTRIAL APPLICABILITY The present invention provides a method for efficiently searching for a substance that suppresses cat urine odor using the response of an olfactory receptor as an index.

The present inventors have discovered that thiols such as 3-mercapto-3-methylbutylformate (MMF), 3-mercapto-3-methyl-1-butanol (MMB), etc., which are contained in cat urine and contribute to feline urine odors. We succeeded in identifying a new olfactory receptor that responds to the genus. In addition, the present inventor evaluates and selects a substance that suppresses cat urine odor by suppressing odors through olfactory receptor antagonism by using the response of the olfactory receptor or a polypeptide having a similar function as an index. I found that it is possible.

Accordingly, the present invention is a method for searching for cat urine odor inhibitors, comprising the following:
adding a test substance and a cat urine odor-causing substance to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions;
measuring the response of the olfactory receptor polypeptide to the causative agent of cat urine odor; and
identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response;
to provide a method comprising:

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to efficiently search for a cat urine odor inhibitor capable of selectively deodorizing cat urine odor by inhibiting odors by olfactory receptor antagonism.

Response of human olfactory receptors to causative agents of cat urine odor. A: MMF, B: MMB. The horizontal axis indicates individual olfactory receptors, and the vertical axis indicates the average value of the response intensity of 2 wells in one experiment. Response of OR2M3 to various concentrations of cat urine odor causative agents. A: response to MMF, B: response to MMB. Data are means±SE (n=3 for each). Suppression of OR2M3 responses to MMF (left) and MMB (right) by OR2M3 antagonists. A: acetylcedrene, B: γ-methylionone, C: fluorohydral. The horizontal axis is the antagonist concentration, and the vertical axis is the response intensity. Data are means±SE (n=3). Inhibitory effect on MMF odor and MMB odor by OR2M3 antagonist. A: Odor intensity for MMF odor, B: Odor intensity for MMB odor. AC: acetylcedrene, MIG: γ-methylionone, Flo: Florhydral. All perfumes used as antagonists were used at a concentration of 1% (v/v). White bars indicate odor intensity of reference samples (no OR2M3 antagonist). Data are means±SE (n=3). FIG. 4 is a cross-sectional view of cat urine disposal tool 1 used in Example 4. FIG. Inhibitory effect of cat urine odor by OR2M3 antagonist. AC: Acetylcedrene, Flo: Florhydral, MIG: γ-Methylionone, CWOT: Cedarwood Oil Texas. The bars are the results after 1 day and 7 days from the left. The vertical axis represents the intensity of cat urine odor based on sensory evaluation.

As used herein, the term “odor suppression by olfactory receptor antagonism” refers to the inhibition of receptor response to the target odor molecule by the other odor molecule by applying both the target odor molecule and another odor molecule. , is a means of suppressing odors (including malodours) that are consequently perceived by individuals. Odor suppression by olfactory receptor antagonism is also a means of using other odor molecules, but it is distinguished from means such as air fresheners that negate the target odor with another strong odor (so-called odor masking). be. One example of odor suppression by olfactory receptor antagonism is the use of substances such as antagonists that inhibit the response of olfactory receptors. If a substance that inhibits the response of an odorant molecule that produces a specific odor is applied to the receptor, the receptor's response to the odorant molecule is suppressed, so that the odor that is ultimately perceived by the individual is suppressed. can be done.

As used herein, the term "olfactory receptor polypeptide" refers to an olfactory receptor or a polypeptide having a function equivalent thereto, and a polypeptide having a function equivalent to an olfactory receptor is similar to an olfactory receptor. , which can be expressed on the cell membrane, is activated by the binding of odorant molecules, and when activated, increases the amount of intracellular cAMP by coupling with intracellular Gas and activating adenylate cyclase. (Nat. Neurosci., 2004, 5:263-278).

As used herein, nucleotide and amino acid sequence identities are calculated by the Lipman-Pearson method (Science, 1985, 227:1435-41). Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (Ver.5.1.1; software development), Unit size to compare (ktup) is set to 2 and analysis is performed. Calculated by

As used herein, "at least 80% identity" with respect to nucleotide sequences and amino acid sequences means 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably Identity of 98% or more, preferably 99% or more.

The "cat urine odor" to be suppressed by the present invention is preferably the characteristic urine odor of cat urine immediately after urination (or fresh) and cat urine after urination, preferably contained in cat urine. Cat urine odor caused by sulfur compounds (thiols and sulfides), more preferably 3-mercapto-3-methylbutyl formate (MMF) and / or 3-mercapto-3-methyl-1 - Cat urine odor due to butanol (MMB). MMF and MMB are thiols contained in cat urine, and are known as malodorous components of cat urine (see Non-Patent Documents 1 and 2). These thiols are components peculiar to cat urine, and are main components that bring about cat urine odors that are stronger and have a unique odor compared to dog and human urine odors (Non-Patent Document 1). On the other hand, there is an unpleasant odor that is common to the urine of humans, dogs, and cats, and which is generated after a long time has passed since urination. This unpleasant odor is caused by the ammonia odor caused by the decomposition of urea by urease, and the phenol, p-cresol, Including odors due to indole, etc. However, these unpleasant odors also originate from human and dog urine, and are not characteristic of cat urine. Moreover, since these unpleasant odors are putrid odors caused by substances generated by microbial decomposition of urine components, they are generated from cat urine after urination for some time, and are not generated from cat urine immediately after urination. On the other hand, since the above-mentioned thiol is a component originally contained in cat urine, the odor is generated immediately after urination and continues to be generated from cat urine even after time has passed. Therefore, in order to effectively suppress cat urine odor, it is necessary to suppress thiol odors such as MMF and MMB from urine immediately after urination.

As shown in FIGS. 1 and 2, the present inventor identified OR2M3 among many olfactory receptors as an olfactory receptor that specifically responds to MMF and MMB, which are causative substances of cat urine odor. The olfactory receptor responds to MMF and MMB in a concentration-dependent manner. Therefore, a substance that suppresses the response of OR2M3 or a polypeptide having an equivalent function causes changes in the central recognition of MMF and MMB due to odor suppression by olfactory receptor antagonism, resulting in cat urine odor. can be selectively suppressed.

Therefore, the present invention provides a search method for cat urine odor inhibitors. The method comprises adding a test substance and a cat urine odor-causing substance to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions; measuring the response of the olfactory receptor polypeptide to an odor-causing agent; and identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response. Identified test substances are selected as cat urine odor control agents.

In the method of the present invention, 3-mercapto-3-methylbutylformate (MMF), 3-mercapto-3-methyl-1-butanol (MMB), or a mixture thereof is preferable as the causative agent of cat urine odor. used for Alternatively, cat urine containing MMF or MMB or an extract thereof can also be used as a causative agent of cat urine odor in the method of the present invention. Cat urine may generate ammonia due to the action of microorganisms contained in cat urine when time has passed since urination. or fresh) cat urine, or cat urine that has been sterilized or antibacterially treated.

Another embodiment of the present invention is a method of searching for MMF odor control agents. The method comprises adding a test substance and MMF to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions; measuring the response of the polypeptide; and identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response. In the method, cat urine containing MMF or an extract thereof may be used instead of MMF. Identified test substances are selected as MMF odor inhibitors.

Yet another embodiment of the present invention is a method of searching for MMB odor control agents. The method comprises adding a test substance and MMB to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions; measuring the response of the polypeptide; and identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response. In the method, cat urine containing MMB or an extract thereof may be used instead of MMB. Identified test substances are selected as MMB odor inhibitors.

The method of the present invention described above can be a method performed in vitro or ex vivo. In the method of the present invention, a test substance and the causative substance for cat urine odor are added to the aforementioned olfactory receptor polypeptide responsive to the causative substance for cat urine odor.

The test substance used in the method of the present invention is not particularly limited as long as it is desired to be used as a cat urine odor suppressant. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, or a compound, composition or mixture. good.

The olfactory receptor polypeptide used in the method of the present invention is at least one selected from the group consisting of OR2M3 and polypeptides having functions equivalent thereto, preferably OR2M3. OR2M3 is an olfactory receptor expressed in human olfactory cells. OR2M3 is registered in GenBank under Accession number: KP290147. OR2M3 is a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 encoded by the gene having the nucleotide sequence of SEQ ID NO:1.

Examples of polypeptides having functions equivalent to OR2M3 include polypeptides consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and having responsiveness to MMF and/or MMB. .

Further examples of polypeptides with equivalent function to OR2M3 include olfactory receptor polypeptides from other animals that are homologous to OR2M3. Examples of the olfactory receptor polypeptide homologous to OR2M3 include a mouse- or rat-derived olfactory receptor polypeptide responsive to MMF and/or MMB, such as a mouse olfactory receptor consisting of the amino acid sequence of SEQ ID NO:3. Olfr164 (NP_666662.1), mouse olfactory receptor Olfr165 (NP_666677.1) consisting of the amino acid sequence of SEQ ID NO:4, rat olfactory receptor Olr1570 (NP_001000041.1) consisting of the amino acid sequence of SEQ ID NO:5, and SEQ ID NO:3 A polypeptide consisting of an amino acid sequence that is at least 80% identical to any of the amino acid sequences from 1 to 5 and having responsiveness to MMF and/or MMB.

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 causative agent of cat urine odor. 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; a membrane of an olfactory cell carrying a polypeptide; a recombinant cell or culture thereof genetically engineered to express said olfactory receptor polypeptide; a membrane of said recombinant cell carrying said olfactory receptor polypeptide; It can be used in a form such as an artificial lipid bilayer membrane having the olfactory receptor polypeptide. All of these forms are included within the scope of the olfactory receptor polypeptides used in the present invention.

In a preferred embodiment, the olfactory receptor polypeptide includes cells that naturally express the olfactory receptor polypeptide, such as olfactory cells, or recombinant cells that have been genetically engineered to express the olfactory receptor polypeptide. Cells, or cultures thereof are used. The recombinant cell can be produced by transforming the cell with a vector containing the gene encoding the olfactory receptor polypeptide.

Preferably, in order to promote cell membrane expression of the olfactory receptor polypeptide, a gene encoding an RTP (receptor-transporting protein) is introduced into cells together with a gene encoding the olfactory receptor polypeptide. Preferably, the gene encoding RTP1S is introduced into the cell together with the gene encoding the olfactory receptor polypeptide. Examples of RTP1S include human RTP1S. Human RTP1S is registered in GenBank as Accession number: AY562235, and is a polypeptide consisting of the amino acid sequence of SEQ ID NO:7 encoded by the gene having the nucleotide sequence of SEQ ID NO:6.

According to the method of the present invention, following the addition of the test substance and the cat urine odor-causing substance to the olfactory receptor polypeptide, the response of the olfactory receptor polypeptide to the cat urine odor-causing substance is measured. . The measurement may be performed by any method known in the art as a method for measuring the response of olfactory receptors, such as intracellular cAMP level measurement. For example, it is known that when olfactory receptors are activated by odorants, they couple with intracellular Gas and activate adenylate cyclase, thereby increasing intracellular cAMP levels (Nat. Neurosci., 2004, 5:263-278). Therefore, the response of the olfactory receptor polypeptide can be measured by using the intracellular cAMP level after the addition of the odorant molecule as an indicator. Methods for measuring the amount of cAMP include ELISA, reporter gene assay, and the like. Other methods of measuring olfactory receptor polypeptide responses include calcium imaging methods.

Next, based on the measured response of the olfactory receptor polypeptide, the effect of the test substance on the response to the cat urine odor-causing substance is evaluated, and a test substance that inhibits the response is identified. Evaluation of the action of the test substance is performed by comparing the response of the olfactory receptor polypeptide to which the test substance is added (test substance addition group) to the cat urine odor-causing substance with the response to the cat urine odor-causing substance in the control group. It can be done by As a control group, the olfactory receptor polypeptides to which different concentrations of the test substance were added, the olfactory receptor polypeptides to which no test substance was added, e.g. , the olfactory receptor polypeptide before addition of the test substance, and the like.

For example, the effect of the test substance on the response of the olfactory receptor polypeptide is between the higher concentration test substance addition group and the lower concentration test substance addition group, the test substance addition group and the non-addition group , between the test substance-added group and the control substance-added group, or before and after the addition of the test substance, by comparing the response of the olfactory receptor polypeptide to the cat urine odor-causing substance. When the response of the olfactory receptor polypeptide is suppressed by the addition of the test substance or by the addition of a test substance at a higher concentration, the test substance suppresses the response of the olfactory receptor polypeptide to the cat urine odor-causing substance. It can be identified as an inhibitory substance.

If the response of the olfactory receptor polypeptide in the test substance addition group measured by the above procedure is suppressed compared to the control group, the test substance is added to the olfactory receptor polypeptide against the cat urine odor-causing substance. It can be identified as a substance that suppresses the peptide response. For example, if the olfactory receptor polypeptide response in the test substance addition group is suppressed to preferably 65% or less, more preferably 50% or less, and even more preferably 25% or less compared to the control group, the A test substance can be identified as a substance that inhibits the response of the olfactory receptor polypeptide to the causative agent of cat urine odor. Alternatively, if the response of the olfactory receptor polypeptide in the test substance addition group is statistically significantly suppressed compared to the control group, the test substance is added to the olfactory receptor against the cat urine odor-causing substance. It can be identified as a substance that suppresses the response of body polypeptides.

The test substance identified by the above procedure is a substance capable of suppressing an individual's perception of cat urine odor by suppressing the response of olfactory receptors to the causative agent of cat urine odor. Therefore, test substances identified by the above procedure can be selected as cat urine odor inhibitors. Furthermore, if necessary, the cat urine odor inhibitory action of the test substance identified by the above procedure may be further sensory evaluated. In one embodiment of sensory evaluation in the method of the present invention, the test substance selected by the above procedure is obtained as a candidate substance for suppressing cat urine odor. Next, the cat urine odor inhibitory action of the candidate substance is sensory evaluated. For example, the intensity of the cat urine odor is sensory evaluated in the presence and absence of the candidate substance. As a specific sensory evaluation method, for example, a vial containing a cat urine odor solution and a vial containing a cat urine odor solution and a candidate substance are prepared, and the cat urine odor solution in each vial is evaluated according to the Visual Analogue Scale (VAS). An odor evaluation may be performed. If cat urine odor is suppressed in the presence of the candidate substance, the candidate substance is selected as a cat urine odor inhibitor. Preferably, the odor intensity in the presence of the candidate substance is reduced to 65% or less, more preferably 50% or less, and still more preferably 25% or less compared to the odor intensity in the absence of the candidate substance. , the test substance can be selected as a cat urine odor inhibitor.

The substance selected as the cat urine odor inhibitor by the method of the present invention suppresses the response of the olfactory receptor to the cat urine odor causative agent, thereby suppressing the cat urine odor by suppressing the odor through olfactory receptor antagonism. be able to.

The cat urine odor inhibitor obtained by the present invention can be used as an active ingredient for controlling cat urine odor. For example, the suppressor can be included as an active ingredient for suppressing cat urine odor in a composition or article for suppressing cat urine odor. Alternatively, the inhibitor can be used for the manufacture of a composition or article for inhibiting cat urine odor. Application examples of the cat urine odor inhibitor obtained by the present invention include placing or spraying the inhibitor on cat toilets, cages, houses or beds; Placement or spraying; pet toilets, or toilet sand or sheets containing the inhibitor; pet sheets or paper diapers containing the inhibitor; cleaning agents for pet products containing the inhibitor; deodorant containing; pet toys, clothing, accessories, etc. containing the deodorant; but not limited to these.

Further disclosed herein as exemplary embodiments of the invention are compositions, methods of manufacture, uses or methods. However, the invention is not limited to these embodiments.

[1] A method for searching for a cat urine odor inhibitor, comprising the following:
adding a test substance and a cat urine odor-causing substance to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions;
measuring the response of the olfactory receptor polypeptide to the causative agent of cat urine odor; and
identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response;
method including.
[2] A method for searching for a 3-mercapto-3-methylbutylformate odor inhibitor, comprising the following:
adding a test substance and 3-mercapto-3-methylbutyl formate to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions;
measuring the response of the olfactory receptor polypeptide to 3-mercapto-3-methylbutylformate; and
identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response;
method including.
[3] A method for searching for a 3-mercapto-3-methyl-1-butanol odor inhibitor, comprising the following:
adding a test substance and 3-mercapto-3-methyl-1-butanol to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions;
measuring the response of the olfactory receptor polypeptide to 3-mercapto-3-methyl-1-butanol; and
identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response;
method including.
[4] The causative agent of cat urine odor is
Preferably, 3-mercapto-3-methylbutyl formate, 3-mercapto-3-methyl-1-butanol, mixtures thereof, or 3-mercapto-3-methylbutyl formate or 3-mercapto-3-methyl- Cat urine or its extract containing 1-butanol,
more preferably 3-mercapto-3-methylbutylformate, 3-mercapto-3-methyl-1-butanol, or a mixture thereof,
More preferably, 3-mercapto-3-methylbutylformate or 3-mercapto-3-methyl-1-butanol,
[1] The method described.
[5] Preferably, the method according to any one of [1] to [4], wherein the OR2M3 is a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
[6] Preferably, the polypeptide having a function equivalent to that of OR2M3 is as follows:
consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2, and having responsiveness to 3-mercapto-3-methylbutyl formate and/or 3-mercapto-3-methyl-1-butanol a polypeptide; and
a mouse or rat-derived olfactory receptor polypeptide homologous to OR2M3, responsive to 3-mercapto-3-methylbutylformate and/or 3-mercapto-3-methyl-1-butanol;
At least one selected from the group consisting of
Preferably, the mouse or rat derived olfactory receptor polypeptide homologous to OR2M3 consists of an amino acid sequence that is at least 80% identical to Olfr164, Olfr165, Olr1570, and any of the amino acid sequences of SEQ ID NOS:3-5. At least one selected from the group consisting of polypeptides responsive to mercapto-3-methylbutyl formate and/or 3-mercapto-3-methyl-1-butanol,
The method according to any one of [1] to [5].
[7] preferably further comprising measuring a response to the causative agent of cat urine odor in a control group, and
Preferably, said control group is:
the olfactory receptor polypeptide to which the test substance was not added;
the olfactory receptor polypeptide to which a control substance has been added; or the olfactory receptor polypeptide before the addition of the test substance;
is
The method according to any one of [1] to [6].
[8] Preferably, the following:
When the response of the olfactory receptor polypeptide to which the test substance has been added to the causative substance for cat urine odor is statistically significantly suppressed compared to the response to the causative substance for cat urine odor in the control group , identifying the test substance as a substance that suppresses the response of the olfactory receptor polypeptide to the causative agent of cat urine odor;
The method according to [7], further comprising
[9] Preferably, the following:
The response of the olfactory receptor polypeptide to which the test substance has been added to the causative substance for cat urine odor is preferably 65% or less, more preferably 50% or less, relative to the response to the causative substance for cat urine odor in the control group. more preferably, identifying the test substance as a substance that suppresses the response of the olfactory receptor polypeptide to the causative agent of cat urine odor when the suppression is 25% or less;
The method according to [7], further comprising
[10] Preferably, further comprising selecting a test substance that suppresses the response of the olfactory receptor polypeptide to the cat urine odor causative agent as a cat urine odor inhibitor, [1], [4] to [ 9] The method according to any one of the above items.
[11] Preferably, a test substance that suppresses the response of the olfactory receptor polypeptide to 3-mercapto-3-methylbutyl formate is selected as a 3-mercapto-3-methylbutyl formate odor inhibitor. The method according to any one of [2], [5] to [9], further comprising.
[12] Preferably, a test substance that inhibits the response of the olfactory receptor polypeptide to 3-mercapto-3-methyl-1-butanol is selected as the 3-mercapto-3-methyl-1-butanol odor inhibitor. The method according to any one of [3], [5] to [9], further comprising:
[13] Preferably, any of [1] to [12], wherein the olfactory receptor polypeptide is expressed on a recombinant cell genetically engineered to express the olfactory receptor polypeptide. or the method according to item 1.
[14] The recombinant cell is
Preferably, the cell is introduced with the gene encoding the olfactory receptor polypeptide and the gene encoding RTP1S,
More preferably, it is a cell into which the gene encoding the olfactory receptor polypeptide and the gene encoding human RTP1S have been introduced.
[13] The method described.
[15] Preferably, the recombinant cell of [13] or [14] or its The method according to any one of [1] to [14], wherein a culture is used.
[16] Preferably, the measurement of the response of the olfactory receptor polypeptide is measurement of intracellular cAMP levels by ELISA or reporter gene assay, or measurement by calcium imaging, according to any one of [1] to [15]. the method of.
[17] Preferably, the method according to any one of [1] to [16], further comprising sensory evaluation of the selected test substance.

EXAMPLES Hereinafter, the present invention will be described more specifically by showing Examples.

Cat urine odor-causing substance 3-Mercapto-3-methylbutyl Format (MMF) (Sigma aldrich Co.)
3-Mercapto-3-methy-1-butanol (MMB) (Tokyo Chemical Industry Co., Ltd.)
Test substance Acetylcedrene (methyl cedryl ketone; [1-[(1S,2R,5R,7S)-2,6,6,8-tetramethyltricyclo[5.3.1.0 ^1,5 ]undec-8- en-9-yl]ethanone); (CAS Registry Number: 32388-55-9; Trade Name: Vertofix; International Flavors & Fragrances, Inc.)
γ-methylionone (α-iso-methylionone; 3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one); (CAS Registry Number: 127- 51-5) (International Flavors & Fragrances, Inc.)
Phlorhydral (β-Methyl-3-(1-methylethyl)benzenepropanal); (Registered Trademark (International Registration 0534879)) (CAS Registry Number: 125109-85-5) (Givaudan SA)

Example 1 Identification of olfactory receptors that respond to cat urine odor-causing substances 1) Cloning of human olfactory receptor genes Based on the sequence information registered in GenBank, 417 human olfactory receptors (7 human trace Genes encoding each of the amine-related receptors and five human vomeronasal receptors were cloned. Each gene was cloned by PCR using a human genomic DNA female (G1521: Promega) as a template. Each gene amplified by PCR was integrated downstream of the Flag-Rho tag sequence on the pME18S vector.

2) Preparation of pME18S-human RTP1S vector A gene encoding human RTP1S (SEQ ID NO: 6) was inserted into the EcoRI and XhoI sites of the pME18S vector.

3) Production of Olfactory Receptor-Expressing Cells HEK293 cells expressing 417 human olfactory receptors (including 7 human trace amine-related receptors and 5 human vomeronasal receptors) were produced. A reaction solution having the composition shown in Table 1 was prepared and allowed to stand in a clean bench for 15 minutes. 4.4 μL of the reaction solution was added to each well of a multiwell plate (384-well plate, BioCoat), and then 40 μL of HEK293 cells (2×10 ⁵ cells/cm ² ) were added to seed the cells. Cells were cultured for 24 hours in an incubator at 37° C. and 5% CO ₂ .

4) Luciferase Assay for Olfactory Receptor Response Olfactory receptors expressed in HEK293 cells couple with intracellular Gαs to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP. In this example, a luciferase reporter gene assay was used to monitor the increase in the amount of intracellular cAMP as a luminescence value derived from the firefly luciferase gene (fluc2P-CRE-hygro) to measure the response of cells to the causative agent of cat urine odor. A Renilla luciferase gene fused downstream of the CMV promoter (hRluc-CMV) was simultaneously transfected into cells and used as an internal standard for correcting errors in gene transduction efficiency and cell number. Dual-Glo ^™ luciferase assay system (Promega) was used to measure luciferase activity. After the operation of 3), the medium was removed from the culture of the olfactory receptor-expressing cells, and 30 μL of a medium containing or not containing the cat urine odor-causing substance was added to each well. A DMEM solution containing 300 μM CuCl ₂ was used as the medium. MMF (final concentration: 30 μM) or MMB (final concentration: 100 μM) was used as the causative agent of cat urine odor. Four hours after the addition of the cat urine odor-causing substance, luminescence was measured according to the operation manual of the product, and the value [fLuc/hRluc] was calculated by dividing the firefly luciferase-derived luminescence value by the Renilla luciferase-derived luminescence value. Fold increase was calculated according to the following formula as an index of the responsiveness of the olfactory receptor to the cat urine odor-causing substance stimulus.
Fold increase=X/Y
X: fLuc/hRluc induced by cat urine odor-causing substance stimulation
Y: fLuc/hRluc in the absence of cat urine odor-causing substance stimulation

5) Results The results of analysis by luciferase assay are shown in FIG. As a result of measuring responses to MMF and MMB for 417 types of olfactory receptors, it was confirmed that the olfactory receptor OR2M3 exhibited a high fold increase value to both MMF and MMB. Therefore, the olfactory receptor OR2M3 was considered to be a receptor that responds to both MMF and MMB.

Example 2 Concentration-Dependent Response of OR2M3 to Causative Substance of Cat Urine Odor The olfactory receptor OR2M3, which showed responsiveness to MMF and MMB in Example 1, was analyzed for concentration dependence of its response to MMF and MMB by luciferase assay. First, OR2M3-expressing HEK293 cells were prepared. A reaction solution having the composition shown in Table 2 was prepared and allowed to stand in a clean bench for 15 minutes. 10 μL of the reaction solution was added to each well of a multiwell plate (96-well plate, BioCoat), and then 90 μL of HEK293 cells (2×10 ⁵ cells/cm ² ) were added to seed the cells. Cells were cultured for 24 hours in an incubator at 37° C. and 5% CO ₂ . As a control, cells (mock) that do not express olfactory receptors were prepared. A luciferase assay was performed in the same manner as in Example 1. 75 μL of the medium containing or not containing the causative agent of cat urine odor was added to each well. The final concentration of MMF was adjusted to 0.003-0.03 mM and the final concentration of MMB was adjusted to 0.01-0.1 mM.

The results are shown in FIG. No responses to MMF and MMB were observed in mock cells, whereas concentration-dependent responses to both MMF and MMB were observed in OR2M3-expressing cells. The above results indicate that OR2M3 is an olfactory receptor that recognizes MMF and MMB, which are components of cat urine odor.

Example 3 Suppressive activity of OR2M3 response by acetylcedrene, γ-methylionone, and fluorohydral OR2M3 responses to MMF or MMB in the presence of acetylcedrene, γ-methylionone, or fluorohydral were measured to examine changes in response intensity. . A luciferase assay of OR2M3-expressing HEK293 cells was performed following a similar procedure as in Example 2. The final concentration of MMF was adjusted to 0.03 mM, and the final concentration of MMB was adjusted to 0.1 mM. The final concentrations of acetylcedrene, γ-methylionone and fluorohydral were adjusted in the range of 0-1 mM. fLuc/hRluc values induced by MMF or MMB stimulation alone (X), fLuc/hRluc values in cells not stimulated with MMF or MMB (Y), and MMF or MMB with acetylcedrene, γ-methylionone or The fLuc/hRluc value (Z) induced by co-stimulation with florhydral was determined. The response intensity (Response (%)) of the receptor to MMF or MMB in the presence of acetylcedrene, γ-methylionone, or fluorohydral was determined by the following formula.
Response (%) = (Z-Y) / (X-Y) x 100
Three independent experiments were performed, and the mean value of the response intensity obtained in each experiment was calculated.

The results are shown in FIG. Acetylcedrene, γ-methylionone and furorhydral all inhibited OR2M3 responses to MMF and MMB in a concentration-dependent manner. These results confirmed that these three compounds are OR2M3 antagonists.

Example 4 Ability of OR2M3 antagonists to suppress cat urine odor 1) Ability to suppress MMF odor and MMB odor The OR2M3 antagonists acetylcedrene, γ-methylionone, and florhydral identified in Example 3 suppress MMF odor and MMB odor. Performance was evaluated by sensory tests.

A 0.1% (v/v) solution of MMF or a 1% (v/v) solution of MMB was used as the cat urine odor solution. Antagonist solutions were 1% (v/v) solutions of either acetylcedrene, γ-methylionone, and fluorohydral. Mineral oil was used as the solvent in all solutions. Put two cotton balls in a glass vial (wide-mouth standard bottle No. 11 #85-0611), one cotton ball soaked with 20 μL of either cat urine odor solution, and the other cotton ball with an antagonist. 20 μL of either solution was soaked. The glass vial containing the cotton ball was capped and allowed to stand at 37° C. for 1 hour before being used as a test sample for a sensory test. As a reference sample, a vial impregnated with 20 μL of mineral oil instead of the antagonist solution was prepared.

A sensory test was conducted by three evaluators. Odor evaluation of test samples was performed according to the Visual Analogue Scale (VAS). That is, the evaluator sniffed the sample, and entered the strength of the odor of MMF or MMB at an arbitrary point on the evaluation axis, with the left end being "No odor" and the right end being "Strong odor". The evaluator first rated the odor of the reference sample and then the test sample. The evaluator then evaluated the odor of the reference sample again. The distance from the evaluation point on the evaluation axis of each sample to the left end was calculated as the odor intensity of each sample. Considering the decrease in odor intensity due to olfactory adaptation, the average value of the above two evaluation results was used as the odor intensity of the reference sample. Taking the odor intensity of the reference sample as 100%, the relative value (%) of the odor intensity of each test sample was calculated.

The results of the sensory test are shown in FIG. Acetylcedrene, γ-methylionone and fluorohydral, which showed antagonistic activity of OR2M3 response in Example 3, all suppressed the odor of MMF and MMB.

2) Ability to Suppress Cat Urine Odor Acetylcedrene, γ-methylionone, and fluorohydral were evaluated for their ability to suppress cat urine odor by a sensory test.
[Test Example 1]
As a sample, a sheet-like cat urine disposal tool 1 having a cross-sectional configuration shown in FIG. 5 was manufactured. The cat urine disposal tool 1 comprises an absorbent part 4 consisting of a liquid absorbent material 46 and a superabsorbent polymer 47 sandwiched between them. The absorber portion 4 has a length in the longitudinal direction Y (not shown) of 4 cm, a length in the width direction X of 4 cm, and a length in the thickness direction Z of 0.26 cm. The absorber portion 4 is wrapped with core wrap sheets 44a and 44b from the front side and the back side, and the end of the back side core wrap sheet 44b overlaps the core wrap sheet 44a on the front side. The absorber portion 4 is covered with a top sheet 2 of 6 cm square on the top and a back sheet 3 of 6 cm square on the bottom. The upper, lower, left and right edges of the top sheet 2 and the back sheet 3 of 1 cm are adhered to seal the absorber portion 4 . As each member constituting the cat urine disposal tool 1, the following were used. The antagonist perfume was applied to the core wrap sheet 44a on the surface side so that the amount of the antagonist perfume added was 7% by mass with respect to the weight of the core wrap sheet 44a. A sample manufactured in this manner was referred to as Test Example 1.
Surface sheet 2: 20 g/m ² of polyolefin nonwoven fabric
Back sheet 3: Polyethylene sheet, 20 g/m ²
Front-side core wrap sheet 44a: Tissue paper, 16 g/m ²
Back side core wrap sheet 44b: Tissue paper, 16 g/m ²
Absorbent material 46: Pulp, 100 g/m ²
Super Absorbent Polymer 47: Water absorbent resin made of crosslinked sodium polyacrylate, 117 g/m ²
Antagonist fragrance: Acetylcedrene

[Test Example 2]
Test Example 2 was produced in the same manner as in Test Example 1, except that the antagonist perfume was changed to florhydral.
[Test Example 3]
Test Example 3 was produced in the same manner as in Test Example 1, except that the antagonist perfume was changed to γ-methylionone.

[Comparative Example 1]
Comparative Example 1 was produced in the same procedure as in Test Example 1 except that Cedarwood Oil Texas (CAS registration number: 91722-61-1, trade name: CEDARWOOD OIL TEXAS (manufactured by TEXAROME)) was used instead of the antagonist perfume. bottom.

〔evaluation〕
1 cc of fresh cat urine was dropped into the beaker containing Test Examples 1 to 3 or Comparative Example 1 every day. The beaker after dripping urine was enclosed in a unipack bag. Dropping of cat urine was performed for 7 days. One day and seven days after the start of dripping cat urine, the cat urine odor of Test Examples 1 to 3 and Comparative Example 1 was sensory evaluated according to the following criteria. The sensory evaluation was performed by three full-time panelists, and the odor intensity was determined by discussion. The odor intensity of the cat urine odor after 7 days was set to 5, and evaluation was made according to the following criteria.
5: Strong odor 4: Strong odor 3: Easily perceptible odor 2: Weak odor that makes it possible to identify what it is 1: Barely perceptible odor 0: Odorless

The results are shown in FIG. In Test Examples 1, 2, and 3, cat urine odor was suppressed both 1 day and 7 days after the start of dripping of cat urine as compared with Comparative Example 1.

From the above results, it was shown that the cat urine odor caused by MMF, MMB, etc. is suppressed by the OR2M3 antagonist. Therefore, based on the response of OR2M3, it was clarified that it is possible to efficiently search for a substance that suppresses the odor of cat urine, particularly the thiol odor contained in cat urine.

1 cat urine disposal tool 2 surface sheet 3 back sheet 4 absorbent part 44 core wrap sheet 46 liquid absorbing material 47 super absorbent polymer

Claims (7)

A method for searching for a cat urine odor inhibitor comprising the following:
adding a test substance and a cat urine odor-causing substance to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions;
measuring the response of the olfactory receptor polypeptide to the causative agent of cat urine odor; and
identifying a test substance that inhibits the response of the olfactory receptor polypeptide based on the measured response;
including
The cat urine odor-causing substance is 3-mercapto-3-methylbutyl formate,
Method. 2. The method of claim 1, wherein said OR2M3 is a polypeptide consisting of the amino acid sequence of SEQ ID NO:2. A polypeptide having a function equivalent to that of OR2M3,
a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:2 and having responsiveness to 3-mercapto-3-methylbutylformate; and
a mouse or rat-derived olfactory receptor polypeptide homologous to OR2M3, responsive to 3-mercapto-3-methylbutyl formate ;
The method according to claim 1 or 2, wherein at least one selected from the group consisting of 4. The method of any one of claims 1-3, wherein said olfactory receptor polypeptide is expressed on a recombinant cell that has been genetically engineered to express said olfactory receptor polypeptide. The method according to any one of claims 1 to 4, further comprising measuring the response of the olfactory receptor polypeptide to which the test substance has not been added to the causative agent of cat urine odor. The response of the olfactory receptor polypeptide to which the test substance has been added to the causative agent of cat urine odor is suppressed relative to the response of the olfactory receptor polypeptide to which the test substance has not been added to the causative agent of cat urine odor. 6. The method of claim 5, further comprising identifying said test substance as a substance that inhibits the response of said olfactory receptor polypeptide to said cat urine odor causative agent when tested. The method according to any one of claims 1 to 6 , wherein the measurement of the olfactory receptor polypeptide response is intracellular cAMP level measurement by ELISA or reporter gene assay, or measurement by calcium imaging.

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