Vapor phase stimulation method of olfactory receptor

Abstract

To provide an olfactory receptor response measuring method by vapor phase stimulation with improved throughput and sensitivity.SOLUTION: A method for measuring olfactory receptor response to a volatile substance having volatilized inside a container on a vapor phase includes a), b1) or b2), c) and d) below: a) a process of mounting a cell which expresses an olfactory receptor polypeptide on a container bottom part; b1) a process of inserting a support body into the container from an opening on a top part of the container, holding the body so as not to contact the cell, and then holding a sample including a volatile substance on the support body; b2) a process of inserting the support body including the volatile substance into the container from the opening on the top part of the container, and holding the body so as not to contact the cell; c) a process of exposing the volatile substance on the cell volatilizing from the support body; and d) a process of measuring olfactory receptor response in the cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring an olfactory receptor response to a gas-phase stimulus. [Background technology]

[0002] Substances that volatilize from the odor source are carried by air currents to the nasal cavity. Volatile substances that reach the olfactory epithelium, which is located at the deepest part of the nose, dissolve in a mucus called olfactory mucus. Volatile substances dissolved in the olfactory mucus reach the olfactory nerve cells. Olfactory nerve cells are distributed in the olfactory epithelium and express olfactory receptors (ORs) on their cell membranes.

[0003] Olfactory receptors are a type of seven-transmembrane G protein-coupled receptor that act as odor sensors, and there are approximately 400 types in humans. Some specific olfactory receptors among the 400 types are activated by binding with a specific volatile substance that reaches them, exciting the olfactory nerve cells. Thus, the chemical information of the volatile substance is finally transmitted to the brain as information on "which combination of olfactory receptors has been activated (olfactory receptor code)," and we recognize the strength and characteristics of the odor of the substance.

[0004] Controlling the activity of olfactory receptors, which act as odor sensors in the above-mentioned odor perception process, is used to solve odor problems. To do this, it is necessary to accurately predict which olfactory receptors will be activated in vivo and to what extent. A reporter gene assay is often used as a means of investigating olfactory receptor activity in vitro. In this method, cultured cells expressing olfactory receptors are stimulated by contacting them with a medium in which the target odorant has been dissolved in advance. A gene is introduced into the cells so that luciferase is expressed as a reporter protein when the olfactory receptor is activated. The expressed reporter protein emits light when a substrate is added, and by measuring the degree of luminescence, it is possible to determine whether the target odorant activates the olfactory receptor in question.

[0005] As mentioned above, the currently widely used olfactory receptor response measurement method is the liquid-phase stimulation method, in which a medium in which a specific odorant has been dissolved in advance is dropped onto cells. This method has the advantages of high throughput and easy concentration adjustment. On the other hand, the odors we actually smell are gases containing a wide variety of volatile substances, which may include unidentified substances. With the existing liquid-phase stimulation method in which a specific odorant is dissolved in advance, it is difficult to measure the olfactory receptor response to these unidentified substances. Furthermore, while the volatility of odorants and their solubility in olfactory mucus affect odor perception, it is difficult to take these factors into account with the liquid-phase stimulation method. In addition, applying actual odor-generating samples directly to cells may cause cell damage. Therefore, the conventional liquid-phase stimulation method is insufficient in terms of reproducing accurate olfactory receptor responses in vivo. In order to more closely reproduce the degree of olfactory receptor activity in vivo, it is reasonable to develop a method in which the odor itself (gas) volatilized from the sample to be examined is used to directly stimulate the olfactory receptor, i.e., the olfactory receptor gas-phase stimulation method.

[0006] There have been several reports of olfactory receptor stimulation by gas phase. For example, Non-Patent Document 1 discloses that HEK293 cells expressing mosquito olfactory receptors were spheroidized, and the response of the olfactory receptors was successfully observed by stimulating the cells with an odor gas phase. In addition, Patent Document 1 and Non-Patent Document 2 disclose that the response of the olfactory receptors was successfully measured by performing gas phase stimulation with an odorant filled in the plate reader chamber using GloSenser cAMP Assay. In addition, Non-Patent Document 3 discloses a method of gas phase stimulation by injecting an odor into a sampling bag containing a 96-well plate.

[0007] However, the approach of Non-Patent Document 1 measures olfactory receptor responses using electrodes. This requires precise manipulation and has extremely low throughput. In addition, the approaches of Patent Document 1 and Non-Patent Document 2 are limited to one type of odor stimulus per plate. In addition, for the odorant and olfactory receptor pairs used in such previously reported methods, the gas-phase stimulation method has a sensitivity of only 1 / 100 to 1 / 1000 of that of the liquid-phase stimulation method (Non-Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Patent Publication No. 2018 / 081588 [Non-patent literature]

[0009] [Non-Patent Document 1] Sato, K. & Takeuchi, S. Chemical vapor detection using a reconstituted insect olfactory receptor complex. Angew. Chemie - Int. Ed. 53, 11798-11802 (2014). [Non-Patent Document 2] Kida, H. et al.Vapor detection and discrimination with a panel of odorant receptors. Nat. Commun. 9, (2018). [Non-Patent Document 3] Yosuke Fukutani. "Identification of human olfactory receptors that respond to volatile sulfur compounds by vapor stimulation assay and search for practical odor suppression fragrances." Poster presentation at the 55th Annual Meeting of the Japanese Society of Taste and Smell. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention relates to providing a method for measuring olfactory receptor responses to gas-phase stimuli, which has improved throughput and sensitivity. [Means for solving the problem]

[0011] The inventors have discovered that in a method for measuring olfactory receptor responses using cells, by exposing volatile substances to olfactory receptors via a support inserted into a container in which cells are seeded, an independent gas-phase stimulation environment is formed, enabling receptor responses to be measured in the gas phase with high sensitivity and efficiency.

[0012] That is, the present invention relates to the following. A method for measuring the olfactory receptor response to a volatile substance vaporized in a container in the gas phase, comprising the steps of: a) b1) or b2), c) and d) below: a) placing cells expressing an olfactory receptor polypeptide on the bottom of a container; b1) inserting a support into the container from an opening at the top of the container, holding the support so as not to come into contact with the cells, and then allowing the sample containing a volatile substance to be held on the support; b2) inserting a support containing a volatile substance into the container from an opening at the top of the container and holding the support so as not to come into contact with the cells; c) exposing the cells to volatile substances volatilized from the support; d) measuring olfactory receptor responses in the cells; A method comprising: Effect of the Invention

[0013] According to the method of the present invention, in vivo olfactory receptor activity information in response to a real odor-emitting sample can be reproduced using cells, providing a more accurate and sophisticated technique for controlling olfactory receptor activity than conventional methods. [Brief description of the drawings]

[0014] [Figure 1] Schematic of gas-phase stimulation conditions. A cross-section of one well of a 96-well plate (Corning) is shown. [Diagram 2]Olfactory receptor measurements using the developed gas-phase stimulation system, and the effect of the volume of the stimulation solution on olfactory receptor responses. The horizontal axis shows the None: solvent stimulation, 10 and 100 μM DMDS stimulation, and 30 μM Forskolin stimulation conditions. The vertical axis shows the relative response intensity (fold increase) when the response observed under the None condition is set to 1. Data from three independent experiments are plotted. The bars and errors show the average value and standard error, respectively. Black indicates 100 μL stimulation, and light gray indicates 150 μL stimulation. [Diagram 3] A) Olfactory receptor response signal values ​​for each well of a 96-well plate over a 30-minute period. Acetophenone vapor stimulation was performed at 0 minutes. Rows 1-6 are mock conditions, and rows 7-12 are Olfr145 expression conditions. Luminescence signals were measured every 5 seconds. B) Confirmation of contamination of adjacent wells with stimulants. The vertical axis shows the average sum of luminescence signal values ​​in each well (logarithmic axis). The horizontal axis shows each row of the plate (stimulus concentration) under olfactory receptor expression conditions. Plots and bars show data from a single experiment and the average of three independent experiments, respectively. Asterisks indicate the statistical test results (p<0.05) for each row against row G (Dunnett's multiple comparisons test). [Figure 4] Comparison of sensitivity between the conventional vapor stimulation method and this method. The vertical axis shows the olfactory receptor response value 30 minutes after stimulation. The horizontal axis shows the vapor stimulation method used. The plot and bar show the data from one experiment and the average value of three independent experiments, respectively. [Diagram 5] A) Schematic diagram of the liquid-phase stimulation method and the newly developed gas-phase stimulation method. B) Vapor pressure and LogP value of the odorants used. C-F) Comparison of sensitivity between the liquid-phase stimulation method and the gas-phase stimulation method. The horizontal axis indicates the concentration of each odorant, and the vertical axis indicates the olfactory receptor response (fold increase). Black indicates liquid-phase stimulation, and light gray indicates gas-phase stimulation. The dashed line indicates EC50. ND indicates not determined. The plots are the average values ​​of three independent experiments, and the errors are standard errors. The odorants and olfactory receptors are C) Diacetyl, OR6Y1 mammalian consensus, D) DMDS, OR2T11 primate consensus, E) p-Cresol, OR9Q2, F) AMBROXAN, OR7A17. F) The open circles indicate the stimulation conditions using a cotton ball that had been dripped with an ethanol solution of AMBROXAN and then dried. [Figure 6] A) Time course of the experiment. Saliva samples were collected at 9 points over 2 days starting immediately after waking up. B) Odor intensity of saliva samples. Sensory evaluation data from one person. C) DMDS concentration (μM) in the saliva headspace. D) Response of OR2T11 primate consensus to gas-phase stimulation with saliva. B-D) The horizontal columns show saliva samples at each time point, connected by dashed lines. n=1. [Figure 7] A) Time course of the experiment. B) Response of OR2T11 primate consensus to vapor-phase stimulation with putrefied urine. n=1. [Figure 8] A) Time course of the experiment. B) Conditions for each sample. Sample 1 is the cotton ball itself, while samples 2-4 differ in whether detergent or fabric softener was used during washing. Sample 2 was washed with tap water only, sample 3 was washed with laundry detergent, and sample 4 was washed with laundry detergent and fabric softener. C) Odor intensity of each cotton ball sample. Mean + standard error of sensory data from three investigators. D) Measurement results of AMBROXAN volatilized from each cotton ball. Extracted ion chromatogram of m / z=221. E) OR7A17 mammalian consensus response to gas-phase stimulation using each cotton ball sample. n=1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The method for measuring olfactory receptor responses of the present invention is a method for measuring, in the gas phase, olfactory receptor responses to volatile substances vaporized in a container, and includes the following steps a), b1) or b2), c) and d). a) placing cells expressing an olfactory receptor polypeptide on the bottom of a container; b1) inserting a support into the container from an opening at the top of the container, holding the support so as not to come into contact with the cells, and then allowing the sample containing a volatile substance to be held on the support; b2) inserting a support containing a volatile substance into the container from an opening at the top of the container and holding the support so as not to come into contact with the cells; c) exposing the cells to volatile substances volatilized from the support; d) measuring olfactory receptor responses in the cells;

[0016] The container used in the method of the present invention may be any container that has an opening at the top and on which cultured cells can be placed, and examples thereof include cell culture plates such as multi-well (1536-well, 384-well, 96-well, 48-well, 24-well, 12-well, 6-well, etc.) microplates, multi-well (8-well, 4-well, 2-well, etc.) chambers, flask-type culture containers, tubes, etc. Among these, multi-well microplates are preferred in that multiple samples can be measured simultaneously, and 384-well, 96-well, and 48-well multi-well microplates are preferred in terms of ease of insertion of supports and operability. The material of the culture vessel is not particularly limited and can be appropriately selected from known culture vessel materials depending on the purpose. Examples of the material include glass, polystyrene, polypropylene, and polycarbonate.

[0017] A culture of cells expressing an olfactory receptor polypeptide is placed on the bottom of the container (step a). An olfactory receptor polypeptide refers to an olfactory receptor or a polypeptide having a function equivalent thereto, and a "polypeptide having a function similar to that of an olfactory receptor" refers to a polypeptide that, like the olfactory receptor, is activated by the binding of odor molecules and, when activated, has the function of increasing the amount of intracellular cAMP by coupling with Gαs or Gαolf in the cell to activate adenylate cyclase. The olfactory receptor polypeptide used in the present invention may be any olfactory receptor polypeptide derived from a mammal, such as primates including humans and chimpanzees, or rodents including mice and rats, and more preferred examples include the 400 or more olfactory receptors found in humans and polypeptides having equivalent functions. Information on human, mouse and rat olfactory receptors can be obtained from GenBank [www.ncbi.nlm.nih.gov]. Furthermore, polypeptides having functions similar to those of the olfactory receptors of the present invention include polypeptides that are at least 90%, preferably 95% or more, and more preferably 99% or more identical in amino acid sequence to the olfactory receptor polypeptide and have functions equivalent to those of the olfactory receptor. In addition, polypeptides having functions similar to those of olfactory receptors include, for example, consensus olfactory receptor polypeptides (Patent Application No. 2022-021606) in which amino acids that are highly common between homologous genes of olfactory receptors of mammals other than humans have been introduced into the human olfactory receptor.

[0018] Examples of cells expressing olfactory receptor polypeptides include cells that naturally express olfactory receptor polypeptides, or recombinant cells genetically engineered to express olfactory receptor polypeptides, preferably recombinant human cells genetically engineered to express human olfactory receptor polypeptides. Examples of host cells that can be used include human embryonic kidney (HEK293) cells, Chinese Hamster Ovary (CHO) cells, Xenopus oocytes, COS, yeast, bacteria, and cells derived from the olfactory epithelium.

[0019] The recombinant cell can be prepared by transforming a cell with a vector incorporating a gene encoding an olfactory receptor polypeptide. Alternatively, the olfactory receptor polypeptide can be expressed by directly introducing a transcription product of the gene into the cell. Preferably, in order to promote the cell membrane expression of the olfactory receptor polypeptide, a gene encoding an RTP (receptor-transporting protein) is introduced into the cell together with the gene encoding the olfactory receptor polypeptide. Preferably, a gene encoding RTP1S is introduced into the cell together with the gene encoding the olfactory receptor polypeptide. An example of RTP1S is human RTP1S. Human RTP1S is a protein registered in GenBank as GI:50234917.

[0020] The nature of the cells to be placed is not particularly limited, and the cells may be in either a suspended or adhered state, for example, a cell gel mixture, a sheet-like cell culture, a cell mass including spheres, a cell suspension, etc.

[0021] Next, a support is inserted into the container through the opening at the top of the container and held so as not to come into contact with the cells, and then a sample containing a volatile substance is held on the support (step b1), or a support containing a volatile substance is inserted into the container through the opening at the top of the container and held so as not to come into contact with the cells (step b2). The support is capable of holding a sample containing a volatile substance inside or on its surface. The support is made of a moldable material that is held midway through the container so that it abuts against the container wall when inserted from the container opening and does not come into contact with the cells placed in the previous step, and the material and shape of the support are not particularly limited.

[0022] Suitable examples of the support include liquid-retaining (absorbent) supports such as fiber materials and flexible porous bodies such as sponges. Examples of the fiber material include natural cellulose fibers such as pulp and cotton, regenerated cellulose fibers such as rayon, and synthetic fibers such as polyethylene, polypropylene, polyester, polyamide, and polyacrylate. Particularly preferred fibers with high water absorption include natural cellulose fibers such as pulp and cotton, regenerated cellulose fibers such as rayon, and polyacrylate fibers. Examples of supports for holding solid samples include well inserts for microplates made of membranes such as polyester or polycarbonate, and metal meshes such as stainless steel.

[0023] When a fiber material is used as the support, the fiber material is preferably in the form of a mass or a molded body. Examples of the mass include cotton balls and cotton plugs. Examples of the molded body include nonwoven fabrics and paper, three-dimensional molded products of the nonwoven fabrics and paper, pulp molded products, woven fabrics, three-dimensional molded products of the woven fabrics, and knitted products knitted in two or three dimensions. Of these, it is preferable to use a cotton ball from the viewpoint of ease of use.

[0024] The sample containing a volatile substance can be retained on the support by either inserting the support into a container and retaining it therein, and then allowing the sample containing a volatile substance to be retained on the support (step b1), or by inserting a support on which a sample containing a volatile substance has already been retained (a support containing a volatile substance) into the container and retaining it therein (step b2). The support is inserted into the container from the opening at the top, and is held in place so as not to come into contact with the cells. Figure 1 shows a schematic diagram of the support (cotton ball) inserted. As shown in Figure 1, a support (cotton ball) of an appropriate size is selected to match the size of the container, and it is inserted from the opening at the top of the container so as to be held midway through the container without coming into contact with the cells.

[0025] A sample containing a volatile substance is a volatile substance (odor substance) for which an olfactory receptor response is evaluated in the present invention, or an actual sample that emits the odor. The volatile substance is not particularly limited as long as it is a substance (odor substance) that acts on olfactory receptors to cause the perception of an odor, and may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, or a compound, composition, or mixture, and may be a liquid or a solid.

[0026] An actual sample that emits an odor is a sample that contains the above-mentioned odorous substances, and examples of such samples include body fluids (e.g., saliva, urine), wastewater, as well as samples that contain odorous substances derived from food, fragrances, cosmetics, pharmaceuticals, cleaning agents, daily necessities, and laundry.

[0027] A sample containing a volatile substance can be prepared by dissolving a volatile substance (odor substance) or an actual sample that emits the odor in question in an appropriate solvent, either directly or after concentrating it. The solvent is preferably a non-volatile solvent, or in the case of a volatile solvent, a solvent that does not cause cytotoxicity, such as mineral oil, water, and the like. In cases where the evaporated solvent may cause cytotoxicity, it is preferable to thoroughly evaporate the solvent from the support before using it to stimulate the cells with an odor. Examples of such solvents include ethanol and acetone.

[0028] When the sample is in the form of a solution, it may be held on the support by injecting it using a pipette, an automatic dispenser, etc. The injection amount may be appropriately set depending on the number of plate wells and the type of support, but for example, when a 96-well plate and a cotton ball are used, it is sufficient if it is less than 200 μL, preferably 100 μL or more and less than 150 μL, and more preferably 150 μL.

[0029] The volatile substances volatilized from the support are then exposed to cells expressing an olfactory receptor polypeptide (step c). Volatile substances volatilizing from the support can be exposed to cells expressing an olfactory receptor polypeptide by contacting the volatile substance with a cell culture medium containing the olfactory receptor polypeptide or the olfactory receptor polypeptide in the gas phase, and the temperature and time can be set appropriately depending on the concentration of the volatile substance, the measurement method, etc. The temperature can be set to, for example, 37°C when using Dual-Glo™ luciferase assay system (Promega). When using GloSenser cAMP Assay (Promega), the temperature can be set to 20°C to 37°C, but when using 22F plasmid, the temperature is preferably 20°C to 25°C, and more preferably 25°C from the viewpoint of responsiveness. When using 20F plasmid in GloSenser cAMP Assay (Promega), the temperature is preferably 30°C to 37°C, and more preferably 37°C from the viewpoint of responsiveness. For example, when the reporter gene assay is used, the time can be 2 to 4 hours, preferably 3.5 to 4 hours, when using the Dual-Glo™ luciferase assay system (Promega). When using the GloSenser cAMP Assay (Promega), which allows measurement over time, the time is less than 1 hour, preferably 30 minutes.

[0030] The measurement of the olfactory receptor response (step d) may be carried out by any method used in the art. It is known that when an olfactory receptor is activated by an odor molecule, it couples with a Gαs type G protein (e.g., Gαs, Gαolf) in the cell to activate adenylate cyclase, thereby increasing the amount of intracellular cAMP. Therefore, the response of the olfactory receptor polypeptide to the causative substance can be measured by using the amount of intracellular cAMP after the addition of the causative substance as an index. Methods for measuring the amount of cAMP include ELISA and reporter gene assays. Another method for measuring the response of the olfactory receptor polypeptide includes calcium imaging. When the olfactory receptor is co-expressed with a Gαq type G protein (Gα15) that can couple with various receptors, the olfactory receptor bound to the ligand increases the calcium ion concentration in the cell. Another method includes measurement by electrophysiological techniques. In electrophysiological measurements, for example, cells (Xenopus oocytes, etc.) in which the olfactory receptor polypeptide is co-expressed with other ion channels are prepared, and the activity of the ion channel on the cell is measured by patch clamping, two-electrode voltage clamping, or the like, to measure the response of the olfactory receptor polypeptide. Among these, in terms of sensitivity and throughput, it is preferable to measure the response of the olfactory receptor by a reporter gene assay method using a luminescent substance such as luciferase. EXAMPLES

[0031] The present invention will be described more specifically below with reference to examples. Reference Example 1 Preparation of olfactory receptor expressing cells 1) Cloning of olfactory receptor genes For the primate consensus of OR2T11, a phylogenetic tree analysis was performed on the homologous genes obtained by BLAST search with the amino acid sequence of human OR2T11 (NP_001001964.1) as the query sequence and the name of the organism to be searched as primate. A clade was selected that included human OR2T11, covered many OR2T11s from other organisms, and contained as few genes other than OR2T11 as possible. Of the total 16 genes contained in that clade, 15 genes excluding human OR2T11 were identified as primate orthologs. Next, alignment analysis and identification of consensus amino acids were performed on the amino acid sequences of a total of 16 genes including human OR2T11 as described below. For the mammalian consensus of OR7A17, the amino acid sequence of human OR7A17 (NP_112163.1) was used as the query sequence, and 145 genes with OR7A17 in their names were identified as orthologs from the top 250 homologous genes searched by BLAST. The amino acid sequences of these 145 genes and human OR7A17 were added to the total of 146 genes, and alignment analysis and identification of consensus amino acids were performed as described below. Alignment analysis of the identified gene group was performed using ClustalW, and further adjustments were made to optimize the amino acids or amino acid motifs that are highly conserved among olfactory receptors. Ballesteros-Weinstein residue numbering was assigned with reference to the results of aligning all mouse olfactory receptors shown in the literature (Ikegami K et al. PNAS 117:2957-2967 (2020)). Based on the alignment results, a consensus olfactory receptor was designed using Jalview. In the alignment, when there is one amino acid residue that is different from the amino acid residue of the reference amino acid sequence and has an occurrence frequency of 50% or more at a position corresponding to each amino acid position of the original human olfactory receptor amino acid sequence serving as a reference, the amino acid residue of the reference amino acid sequence was modified to that amino acid residue. In addition, even if there was one amino acid residue different from the amino acid residues in the reference amino acid sequence and with an occurrence frequency of 50% at a position corresponding to each amino acid position in the original reference human olfactory receptor amino acid sequence, if the occurrence frequency of the amino acid residue in the reference amino acid sequence was also 50%, the amino acid residues in the reference amino acid sequence were not modified. On the other hand, if an amino acid with an occurrence frequency of 60% or more was present at a position corresponding to a deletion position in the original reference human olfactory receptor amino acid sequence, the most conservative amino acid was inserted at the deletion position of the reference amino acid sequence. The topology of the olfactory receptor in the design was confirmed using TMHMM (Transmembrane Hidden Markov Model). The DNA sequences (SEQ ID NO: 1 and SEQ ID NO: 3, respectively) encoding the designed olfactory receptor polypeptides (OR7A17 consensus (SEQ ID NO: 2) and OR2T11 consensus (SEQ ID NO: 4)) were obtained by DNA synthesis after optimizing the base sequence codons corresponding to the amino acid sequence for expression in human cultured cells. EcoRI and XhoI sites were added to both ends of this base sequence, and it was recombined into the EcoRI and XhoI sites created downstream of the Flag-Rho tag sequence on the pME18S vector.

[0032] 2) Construction of pME18S-human RTP1S vector The gene encoding human RTP1S, which transports olfactory receptor proteins produced in cultured cells to the cell membrane surface, was inserted into the EcoRI and XhoI sites of another pME18S vector.

[0033] 3) Creation of olfactory receptor expressing cells HEK293 cells expressing the above olfactory receptors were prepared. The reaction solution with the composition shown in Table 1 or Table 2 was prepared and left to stand in a clean bench for 15 minutes, and then added to each well of a 96-well plate (BD). Next, 100 μL of HEK293 cells suspended in DMEM (Nacalai) were added to each well at 2 × 10 5 cells / cm 2 The cells were seeded at 100°C and cultured for 24 or 48 hours in an incubator maintained at 37°C and 5% CO2. As a control, cells that do not express olfactory receptors (mock) were prepared by transfecting an empty vector that did not incorporate the olfactory receptor gene.

[0034] [Table 1]

[0035] [Table 2]

[0036] Reference Example 2 Reporter gene assay (Dual-Glo™ luciferase assay system) Olfactory receptors expressed in HEK293 cells increase the amount of intracellular cAMP by coupling with endogenous Gαs and activating adenylate cyclase. In this study, a luciferase reporter gene assay was used to measure odor responses, which monitors the increase in intracellular cAMP as the luminescence value derived from the firefly luciferase gene (luc2P-CRE-Hygro). In addition, a Renilla luciferase gene fused downstream of the CMV promoter (Rluc-CMV) was simultaneously transfected and used as an internal standard to correct errors in gene transfection efficiency and cell number. Luciferase activity was measured using the Dual-Glo™ luciferase assay system (Promega) according to the product's operating manual. For various stimulation conditions, the luminescence value derived from firefly luciferase was divided by the luminescence value derived from Renilla luciferase to calculate fLuc / Rluc. The fold increase was calculated by dividing the fLuc / Rluc induced by odorant stimulation by the fLuc / Rluc in cells not stimulated with the odorant, and used as an index of response strength. Dose-response curve analysis was performed using GraphPad Prism (8.3.0). EC50 was calculated using nonlinear regression (three parameters).

[0037] Reference Example 3 GloSenser™ cAMP Assay Olfactory receptors expressed in HEK293 cells increase the amount of intracellular cAMP by coupling with endogenous Gαs and activating adenylate cyclase. In this study, the real-time olfactory receptor response was measured using the GloSenser™ cAMP Assay, which monitors the increase in intracellular cAMP as luminescence values ​​derived from a modified firefly luciferase gene. Luciferase activity was measured using the GloSensor™ cAMP Reagent, following the product's operating manual. EnSight (PerkinElmer) or FDSS (Hamamatsu Photonics) was used as the luminescence measurement device.

[0038] Example 1: Development of a vapor-phase stimulation method for olfactory receptors The olfactory receptor plasmid produced according to Reference Example 1 was transfected into HEK293 cells in a 96-well plate (manufactured by Corning or Grainer) to prepare HEK293 cells expressing olfactory receptor proteins. The entire medium was removed, and 35 μL of new medium (DMEM) was added. Then, cotton balls (manufactured by Osaki Medical Co., Ltd. (31420)) were packed into the upper part of each well using tweezers (Figure 1). At this time, it was visually confirmed from the bottom of the plate that the cotton balls were not attached to the bottom of the wells. Then, an odorant (0 μM, 10 μM, or 100 μM) dissolved in DMEM was added to stimulate the olfactory receptor with the odorant. Dimethyl disulfide (DMDS) was used as the odorant, and OR2T11 (OR2T11 primate consensus), which responds to DMDS, was used as the olfactory receptor. The amount of the stimulating solution was 100μL or 150μL because 200μL may exceed the water retention capacity of the cotton ball. After the odor stimulation, the plate was sealed and left to stand for 3 to 4 hours under conditions of 37℃ and 5% CO2. After standing, the plate seal was removed and the cotton balls packed in the wells were removed with tweezers. The cell culture medium was completely removed using a pipette. Then, 75μL of new culture medium (DMEM) was added to each well, and olfactory receptor activity was measured according to Reference Example 2. Forskolin, a non-volatile substance that activates adenylate cyclase, was used as a stimulating sample as a control condition. Forskolin was adjusted to 30μM with DMEM. If luminescence is observed under the control condition, it can be determined that the stimulating solution soaked in the cotton ball is mixed into the cell solution, which is a liquid-phase stimulation, and if no luminescence is observed, it can be determined that a gas-phase stimulation has been established.

[0039] No clear luminescence signal was observed when the forskolin solution was applied to the gas phase. In other words, the establishment of gas phase stimulation was confirmed. In addition, the OR2T11 response was observed in a DMDS concentration-dependent manner (Figure 2). Furthermore, it was found that a stimulation solution volume of 150 μL could induce a stronger olfactory receptor response than a volume of 100 μL (Figure 2).

[0040] Example 2 Confirmation of independent gas phase environment The influence of odorant contamination on adjacent wells was examined. Olfactory receptor expressing cells were prepared according to the method described in Reference Example 1, and the olfactory receptor response to odor stimulation was measured according to the method described in Reference Example 3. The composition of Table 2 was used for the preparation of olfactory receptor expressing cells. The olfactory receptor plasmid produced according to Reference Example 1 was transfected into HEK293 cells in a 96-well plate (manufactured by Corning or Grainer) to prepare HEK293 cells expressing olfactory receptor proteins. The cell solution was then replaced with 35 μL of GloSensor™ cAMP Reagent solution adjusted to 4% (v / v) with DMEM. Then, cotton balls (manufactured by Osaki Medical Co., Ltd. (31420)) were packed into the upper part of each well using tweezers (Figure 1). The plate was sealed and left to stand at 37 ° C for 2 hours. After standing, the plate seal was removed and inserted into an FDSS chamber maintained at 25 ° C, and the olfactory receptor response was measured according to Reference Example 3. After automatically dispensing 150 μL of the stimulation solution, luminescence measurement was performed for 30 minutes. At this time, when a 100 ppm odorant solution was used as the stimulation solution, we investigated whether the olfactory receptors expressed in the adjacent wells would respond. The odorants (olfactory receptors) used were Acetophenone (Olfr145), Heptanal (Olfr2), and Eugenol (Olfr73).

[0041] The results are shown in Figure 3. A response was induced in the wells stimulated with 1% acetophenone, confirming that gas-phase stimulation of the olfactory receptors was established (Fig. 3A). However, no clear olfactory receptor response was induced in the adjacent wells (Fig. 3A, B). When the rate of contamination was estimated by comparing the receptor response in the adjacent wells with the different dose-dependent receptor responses, it was found that only about 0.1% of the adjacent wells were contaminated (Fig. 3B). In a typical olfactory receptor reporter gene assay, the dynamic range of the olfactory receptor response is 10 3(Mainland, JD, Li, YR, Zhou, T., Liu, WL in. L. & Matsunami, H. Human olfactory receptor responses to odorants. Sci. data 2, 150002 (2015)). Taking the above into consideration, it was found that in the olfactory receptor reporter gene assay, each well can be considered as an independent gas-phase stimulus environment. In fact, it was possible to observe concentration-dependent olfactory receptor responses using the same plate (Figure 3B).

[0042] Example 3: Verification of the sensitivity of the receptor response measurement system The sensitivity of the olfactory receptor response measurement system using the gas-phase stimulation system of the present invention was compared with that of the method disclosed in Non-Patent Document 2. In the gas-phase stimulation method disclosed in Non-Patent Document 2, a 96-well plate containing 25 μL of stimulation solution in each well is left in the plate reader chamber for 5 minutes before measuring the olfactory receptor response, so that the plate reader chamber is filled with odorants. Then, the measurement plate is immediately inserted. The cells in the wells are gas-phase stimulated with odorants volatilized from the stimulation solution injected between each well of the measurement plate. In this example, some of the combinations of odorants and olfactory receptors disclosed in Non-Patent Document 2, that is, the odorants (olfactory receptors) are Acetophenone (Olfr145), Heptanal (Olfr2), and Eugenol (Olfr73), were used to compare the olfactory receptor response obtained by the method of Non-Patent Document 2 with the olfactory receptor response obtained by the method of the present invention. In both cases, the olfactory receptor response was observed according to Reference Example 3.

[0043] As a result, the present invention showed equal or higher response than the conventional method for all odor substances used (Figure 4). In particular, a more pronounced response was obtained with Eugenol, which has the lowest vapor pressure of the three substances used, than with the conventional method. The above results indicate that the present method, due to its small gas phase volume, increases the gas phase concentration more efficiently than previously disclosed olfactory receptor gas phase stimulation methods. Using this method, it is possible to detect olfactory receptor responses to odor substances with a wider range of chemical properties than conventional methods with high sensitivity.

[0044] Example 4 Sensitivity of receptor response measurement system (comparison with liquid-phase stimulation) The sensitivity of the gas-phase stimulation method was examined in comparison with the liquid-phase stimulation method (FIG. 5A). In the gas-phase stimulation method, it is expected that the volatility from the cotton ball and the solubility in the cell culture medium are involved in the olfactory receptor response. Therefore, four odorants with significantly different vapor pressures (VP) and octanol / water partition coefficients (LogP) were prepared from among odorants for which olfactory receptors have been identified (FIG. 5B). The amount of the stimulation solution was 150 μL, and the olfactory receptor was stimulated in the gas phase in the same manner as in Example 1 otherwise. The resulting olfactory receptor response was measured according to Reference Example 2. Regarding the liquid-phase stimulation method, after preparing HEK293 cells expressing olfactory receptor proteins in the same manner as in Example 1, the entire medium was removed and 75 μL of a stimulation solution in which the odorant was dissolved in DMEM was added. After the odor stimulation, the plate was sealed and left to stand for 3 to 4 hours under conditions of 37°C and 5% CO2. After standing, the olfactory receptor response was measured according to Reference Example 2.

[0045] As a result, for Diacetyl and DMDS (dimethyl disulfide), which have high vapor pressures and low LogP values ​​among the four substances, the gas-phase stimulation method was more sensitive than the liquid-phase stimulation method, while for p-Cresol, which has intermediate vapor pressures and LogP values, the gas-phase stimulation method showed the same sensitivity as the liquid-phase stimulation method (Figure 5C-E). On the other hand, no response was observed for AMBROXAN, which has low vapor pressure and high LogP. It is considered that the amount of AMBROXAN that evaporates into the gap in the well, which is low volatile and difficult to dissolve in water, is small, and the amount of AMBROXAN dissolved in the cell solution is so small that it does not cause an olfactory receptor response. However, when the solvent was changed from water to ethanol and a cotton ball was dropped on the cotton ball and the ethanol was evaporated for 1 hour, an olfactory receptor response was observed in the same concentration range as the liquid-phase stimulation (Figure 5F). Thus, it was shown that by using this gas-phase stimulation method, it is possible to measure the olfactory receptor response for all odorants used in this study on the same concentration scale as the liquid-phase stimulation.

[0046] Example 5 Olfactory receptor responses to salivary volatiles By using the methods described in Examples 1, 2, and 3, the odor volatilizing from a real sample can be detected as an olfactory receptor response. In this example, we performed detection of odors volatilized from saliva. If saliva is applied directly to cells, either as is or after ultrafiltration, it will cause cell death. The gas-phase stimulation system is also excellent for detecting odors volatilized from real samples because it does not induce cell death. It is known that bad breath originates mainly from within the oral cavity (Shibuya Koji. Research into the components and origins of physiological bad breath. Journal of Oral Hygiene 51, 778-792 (2001)). The oral cavity is covered with saliva. Therefore, it is thought that odors volatilizing from saliva account for much of bad breath. Measuring olfactory receptor activity in response to salivary volatile substances can be said to be a measurement of the degree of olfactory receptor response to the complex odor known as bad breath. This will provide a starting point for sensory deodorization of bad breath by targeting olfactory receptors.

[0047] Therefore, olfactory receptor stimulation with saliva was performed (Figure 6A). For the olfactory receptor, a primate consensus of OR2T11, which is known to receive many sulfur-containing volatile compounds including DMDS, one of the substances that cause bad breath, was used. For saliva collected at each time after waking up, odor intensity evaluation, measurement of the concentration of the DMDS component contained in the bad breath, and measurement of the degree of activation of OR2T11 were performed. The amount of saliva used for gas-phase stimulation of the olfactory receptor was 150 μL. Other preparations of olfactory receptor-expressing cells and the method of gas-phase stimulation were the same as in Example 1. The obtained olfactory receptor response was measured according to Reference Example 2. The odor intensity evaluation was performed by one tester before the measurement of the olfactory receptor response and before the measurement of the DMDS content. For the DMDS concentration measurement, the saliva headspace was aerated through a Tenax adsorption tube to collect saliva volatile substances. The collected sample was subjected to TDU-GC-MS and DMDS was measured. The estimated DMDS concentration was calculated from the calibration curve.

[0048] The results of the experiment are shown in Figure 6. A clear olfactory receptor response was observed only when the gas-phase stimulation was performed using morning saliva, which has a strong odor and contains a high concentration of DMDS (Figure 6B-D). In other words, it was shown that the use of this gas-phase stimulation system makes it possible to detect olfactory receptor responses that reflect the odor intensity of salivary volatile substances and the concentration of oral odorants.

[0049] Example 6 Olfactory receptor response to putrefactive urinary volatiles In addition to bad breath, putrid urine odor is also a problem faced in nursing and childcare settings, and several substances that cause it have been identified. For example, alcohols, phenols, ketones, aldehydes, pyrroles, and sulfur-containing volatile compounds have been identified (Wagenstaller, M. & Buettner, A. Quantitative Determination of Common Urinary Odorants and Their Glucuronide Conjugates in Human Urine. Metabolites3, 637-657 (2013).). Therefore, focusing on sulfur-containing volatile compounds, we examined whether putrid urine activates olfactory receptor polypeptides. Urine samples were collected the day before the olfactory receptor response measurement. They were then immediately filter-sterilized. Next, a urine sample that emits a putrid urine-like odor was prepared by treating it overnight at 37°C. Similarly, a urine sample that was not filter-sterilized was also prepared. The amount of urine sample used for stimulation was 150 μL. The olfactory receptor polypeptide used was the primate consensus of OR2T11, which is known to perceive many sulfur-containing volatile compounds. The preparation of olfactory receptor-expressing cells and the gas-phase stimulation method were the same as in Example 1. The resulting olfactory receptor response was measured according to Reference Example 2.

[0050] As a result, receptor responses were observed in all urine samples, and the response intensity was higher in the unfiltered samples than in the filter-sterilized samples (Figure 7B). This suggests the involvement of microorganisms in the generation of OR2T11 agonists in urine, and demonstrates that the degree of olfactory receptor activity in response to the odor of putrid urine can be observed.

[0051] Example 7 Olfactory receptor response to washed cotton Using laundry detergent and fabric softener containing AMBROXAN in the fragrance formulation as models, we tested whether a response of OR7A17 activated by AMBROXAN could be obtained when a cotton ball washed with these was subjected to gas-phase stimulation. The cotton ball was washed with laundry detergent and fabric softener containing AMBROXAN on the day before the measurement of olfactory receptor response, and the cotton ball was naturally dried overnight to prepare a sample for gas-phase stimulation. The mammalian consensus of OR7A17, which is known to respond to AMBROXAN, was used as the olfactory receptor. The preparation of olfactory receptor-expressing cells and the method of gas-phase stimulation were the same as in Example 1. The obtained olfactory receptor response was measured according to Reference Example 2. The intensity of the odor volatilized from the cotton ball was evaluated by three test personnel who could smell the odor of AMBROXAN. Regarding the measurement of AMBROXAN volatilized from the washed cotton ball, the Tenax adsorption tube in which the cotton ball headspace was ventilated was subjected to TDU-GC-MS, and (-)-Ambroxide, the main substance of AMBROXAN, was measured. Figure 8A shows the time course of the experiment, and Figure 8B shows the conditions of the cotton ball samples.

[0052] As a result of the experiment, untreated cotton balls (Sample 1) and cotton balls washed with tap water only (Sample 2) did not activate OR7A17, while cotton balls treated with fabric softener (Sample 4) induced the highest receptor response among all samples (Figure 8E). Sample 4 exhibited the strongest sensory scent (Figure 8). In fact, (-)-Ambroxide was also detected in the headspace of the cotton ball (Figure 8D). Thus, it was found that by using the odor volatilized from washed cotton balls as a gas-phase stimulus, it is possible to obtain an olfactory receptor response that describes the odor of washed cotton.

Claims

1. A method for measuring the olfactory receptor response to volatile substances volatilized in a container in the gas phase, comprising the following steps a), b1) or b2), c), and d): a) placing cells expressing an olfactory receptor polypeptide on the bottom of the container; b1) inserting a support into the container through the opening at the upper part of the container, holding it so as not to contact the cells, and then holding a sample containing a volatile substance on the support; b2) inserting a support containing a volatile substance into the container through the opening at the upper part of the container and holding it so as not to contact the cells; c) exposing the cells to the volatile substance volatilized from the support; d) measuring the olfactory receptor response in the cells. A method comprising these steps.

2. The method according to claim 1, wherein the support is a cotton ball.

3. The method according to claim 1, wherein the container is a cell culture plate.

4. The method according to claim 3, wherein the cell culture plate is a multi-well microplate.

5. The method according to claim 1, wherein the response of the olfactory receptor polypeptide is measured by using the amount of cAMP as an index.

6. The method according to any one of claims 1 to 5, wherein the sample containing the volatile substance is a body fluid.

7. The method according to claim 6, wherein the body fluid is saliva.

8. The method according to claim 6, wherein the body fluid is urine.

9. The method according to any one of claims 1 to 5, wherein the support containing the volatile substance is washed cotton fibers.

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