Method of identifying malodor modulating compounds

Abstract

To provide assays that can be used to identify inhibitors, modulators or neutralizers of malodor compounds such as dimethyl trisulfide (DMTS).SOLUTION: Provided herein are polypeptides that bind to the malodor-causing substance DMTS. Also provided are nucleic acid sequences that encode for the polypeptides. Further provided herein is a method for identifying a compound that binds, suppresses, blocks, inhibits, and / or modulates the activity of one or more olfactory receptors that are activated by the malodor-causing substance DMTS, comprising a) contacting the receptor, or a chimera or fragment thereof with the compound and b) determining whether the compound has an effect on the activity of the receptor. Further provided is an expression vector comprising the nucleic acid encoding the polypeptides described as well as a non-human organism or a host cell modified to express a receptor that is activated by DMTS.SELECTED DRAWING: None

Description

Technical Field

[0001] Technical Field of the Invention This technical field relates to assays that can be used to identify odorants and odorant receptors, as well as inhibitors, regulators or neutralizers of odorants and / or aromatic compounds, more specifically malodorous compounds such as dimethyl trisulfide (DMTS).

[0002] Background Olfaction is one of the most complex and least understood of the human sensory systems. There are many steps from the activation of olfactory receptors (ORs) to perception that still require further investigation. If the inventors can understand how the OR code of individual odorants or mixtures is converted into perception, this knowledge can be exploited to bring great benefits in several fields. These fields include odor regulators such as malodor neutralizers that block the perception of unpleasant odors, new flavor and fragrance components to replace non-biodegradable or toxic compounds, and odor enhancers that limit the dependence on compounds that are difficult to supply from natural sources. The combinatorial paradigm of the "olfactory code" is based on the observation that a single OR can be activated by multiple odorants, and conversely, most odorants can activate multiple ORs. The mouse genome has approximately 1,200 distinct intact ORs, in contrast, humans have approximately 400. In both cases, the OR repertoire is activated by thousands of odorants worldwide, and the complexity of this combination allows for the breadth of perceivable olfaction. However, as of 2014, odorants or ligands have been identified for only 82 mouse ORs (about 8%) and 17 human ORs (about 10%) using conventional de-orphanization methods. Furthermore, the physiological relevance of most ligands for human ORs that have been substantially identified in vitro has not been tested at all.

[0003] A method for rapidly and reliably identifying a relatively small subset of ORs within the entire repertoire of ORs present in an organism that is specifically activated or inhibited by one or more odorant substances is described in WO 2014 / 210585. However, when using this method, it is necessary to identify odorant receptors, and more specifically, malodor receptors that are activated by substances that cause specific malodors.

[0004] Compounds that cause malodors, such as dimethyl trisulfide (DMTS), and other closely related polysulfide compounds, such as dimethyl disulfide (DMDS), can generate unpleasant odors that originate, for example, from toilets containing feces and other "toilet" sources or from bad breath. Therefore, there is a desire for a malodor regulator or neutralizer that binds, inhibits, blocks, suppresses, and / or modulates the activity of one or more olfactory receptors that are activated by substances that cause specific malodors, such as DMTS or DMDS. There is a further desire for an assay for identifying new compounds or compound mixtures that bind to such newly identified or newly identified malodor receptors associated with substances that cause specific malodors.

[0005] SUMMARY OF THE INVENTION Provided herein are non-human host organisms or host cells modified to express a receptor that is activated by DMTS and is selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1.

[0006] Furthermore, provided are non-human host organisms or host cells transformed to express a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40.

[0007] Furthermore, provided herein is an expression vector comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40.

[0008] Also provided herein is an expression vector having a nucleic acid comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, or its reverse complement.

[0009] Provided herein are nucleic acids comprising the above nucleic acid sequences and polypeptides comprising the above amino acid sequences.

[0010] Furthermore, the present specification also provides a method for identifying a compound that binds to, inhibits, blocks, suppresses, and / or regulates the activity of at least one olfactory receptor activated by a malodor-causing substance such as dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS), wherein the receptor is a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40, and the method comprises a) contacting the receptor or chimera or fragment thereof with a compound; b) determining whether the compound affects the activity of the receptor. A method is provided that includes the above.

[0011] Also provided is a method for identifying a compound that binds to, inhibits, blocks, suppresses, and / or regulates the activity of at least one olfactory receptor activated by a malodor-causing substance, the method comprising: a. Contacting a test substance and a malodor-causing substance with at least one olfactory receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1; b. Measuring the response of the olfactory receptor to the malodor-causing substance by measuring the response of the olfactory receptor in the presence and absence of the test substance; c. Identifying a test substance that modulates the response of the olfactory receptor based on the responses measured in the presence and absence of the test substance; and d. Selecting the identified test substance as a compound that modulates the response of an olfactory receptor to an odor-causing substance comprising Also provided is a method wherein the odor-causing substance is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

[0012] Furthermore, a method for identifying an odor modulator, comprising a. contacting a test substance and an odor-causing substance with at least one olfactory receptor, wherein the receptor comprises a polypeptide having an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40; b. measuring the response of the olfactory receptor polypeptide to the odor-causing substance; c. identifying a test substance that can suppress the response of the olfactory receptor based on the measured response; and d. selecting a test substance that binds to, suppresses, blocks, inhibits, and / or modulates the response of the olfactory receptor as an odor modulator comprising Also provided is a method wherein the odor-causing substance is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

[0013] Furthermore, provided is a recombinant nucleic acid molecule comprising a nucleic acid containing at least one of a Lucy tag, a FLAG (registered trademark) tag, and / or a Rho tag, and a nucleic acid encoding a receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR4S2, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1 or its reverse complement.

[0014] Still further provided is a non-human host organism or host cell recombinantly modified to express the above nucleic acid or polypeptide.

[0015] Detailed Description of the Invention For the purposes of this specification and the appended claims, the use of "or" means "and / or" unless specifically stated otherwise. Similarly, "comprise", "comprises", "comprising", "include", "includes", and "including" are interchangeable and not intended to be limiting.

[0016] Furthermore, when the description of various embodiments uses the term "comprising", it should be understood that those skilled in the art will be able to alternatively describe the embodiments, in some specific instances, using the terms "consisting essentially of" or "consisting of".

[0017] The following terms have the meanings ascribed to them unless specifically stated otherwise.

[0018] "OR" refers to one or more members of a family of G protein-coupled receptors (GPCRs) that are expressed in olfactory cells. Olfactory receptor cells can also be identified based on morphology or by the expression of proteins that are specifically expressed in olfactory cells. Members of the OR family may have the ability to function as receptors for olfactory signal transduction.

[0019] "DMTS OR" or "DMDS OR" refers to a member of a family of G protein-coupled receptors that are expressed in olfactory cells and that are bound and / or activated by DMTS or DMDS in a binding or activity assay to identify ligands that bind to and / or activate the GPCR. Such assays are described below. The DMTS or DMDS receptors herein will include fragments, variants, as well as chimeric or recombinant nucleic acids or proteins that respond to or bind DMTS or DMDS, including synthetic and naturally occurring ones.

[0020] The "OR" polypeptides are considered to be such because they belong to the seven transmembrane domain G protein-coupled receptor superfamily encoded by a single exon of approximately 1 kb in length and exhibit characteristic olfactory receptor-specific amino acid motifs. The seven domains are called "transmembrane" or "TM" domains TM I - TM VII and are connected by three "intracellular loops" or "IC" domains IC I - IC III and three "extracellular loops" or "EC" domains EC I - EC III. The motifs and their variants are defined by, but not limited to, the MAYDRYVAIC motif (SEQ ID NO: 53) overlapping TM III and IC II, the FSTCSSH motif (SEQ ID NO: 54) overlapping IC III and TM VI, the PMLNPFIY motif (SEQ ID NO: 55) of TM VII, and the three conserved C residues of EC II, and the highly conserved GN residues in TM I [Zhang, X. & Firestein, S. Nat. Neurosci. 5, 124 - 133 (2002); Malnic, B. et al. Proc. Natl. Acad. Sci. U. S. A. 101, 2584 - 2589 (2004)].

[0021] The "OR" nucleic acids encode a family of GPCRs having, for example, seven transmembrane regions with "G protein-coupled receptor activity", which bind to G proteins in response to extracellular stimuli and promote the production of second messengers such as IP3, cAMP, cGMP, Ca 2+ etc.

[0022] "Paralogous" OR genes or "paralogs" are the result of gene duplication and refer to closely related homologous genes within the same species. "Orthologous" OR genes or "orthologs" are defined as being phylogenetically linked by genes present in a common ancestor and refer to closely related homologous genes in other species.

[0023] The "N-terminal domain" region starts from the N-terminus and extends to a region near the start of the first transmembrane region. The "transmembrane region" contains seven "transmembrane domains", which refers to the domain of the OR polypeptide within the plasma membrane and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. The seven transmembrane regions as well as the extracellular and cytoplasmic loops can be identified using standard methods such as hydrophobicity profiles, or as described in Kyte & Doolittle, J. Mol. Biol., 157:105 - 32 (1982), or Stryer. The general secondary and tertiary structures of transmembrane domains, particularly the seven transmembrane domains of G-protein coupled receptors such as olfactory receptors, are known in the art. Thus, the primary structure sequence can be predicted based on known transmembrane domain sequences, as will be described in detail below. These transmembrane domains are useful for in vitro ligand binding assays.

[0024] The phrase "functional effect" in the context of an assay for testing compounds that modulate OR family member-mediated olfactory transduction includes the determination of indirect or direct parameters under the influence of the receptor, such as functional, physical, and chemical effects. This includes ligand binding, changes in ion flux, membrane potential, current flow, transcription, G-protein binding, GPCR phosphorylation or dephosphorylation, signal transducer-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP IP3, or intracellular Ca 2+ ) in vitro, in vivo, and ex vivo, and also includes other physiological effects such as increases or decreases in neurotransmitter or hormone release.

[0025] "Measurement of functional effect" or "confirmation of activity" in the context of an assay means an assay of indirect or direct parameters under the influence of an OR family member, for example, an assay of a compound that increases or decreases functional, physical and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, for example, changes in spectroscopic properties (e.g., fluorescence, absorbance, refractive index), hydrodynamics (e.g., shape), chromatography, or solubility properties, patch clamp, voltage-sensitive dyes, whole cell current, radioisotope efflux, induced markers, oocyte OR gene expression; tissue culture cell OR expression; transcriptional activation of the OR gene or activation-induced genes such as egr-1 or c-fos; ligand binding assay; changes in voltage, membrane potential and conductance; ion flux assay; changes in intracellular second messengers such as cAMP, cGMP, and inositol trisphosphate (IP3); changes in intracellular calcium levels; release of neurotransmitters, etc.

[0026] "Binders", "suppressors", "blockers", "inhibitors", "activators", and / or "modulators" of OR genes or proteins are molecules identified using in vivo, in vitro, and ex vivo assays for olfactory transmission, e.g., ligands, agonists, antagonists, enhancers, and homologs and mimetics thereof, and are used interchangeably to refer to the binding, suppression, blocking, inhibition, inactivation, activation, or modulation of these. Inhibitors are, e.g., compounds that bind, partially or completely block stimulation, decrease, inhibit, prevent, delay activation, inactivate, desensitize, or downregulate olfactory transmission, e.g., antagonists. Activators are, e.g., compounds that bind, stimulate, increase, promote activation, enhance activation, sensitize, or upregulate olfactory transmission, e.g., agonists. Modulators include, e.g., extracellular proteins that bind activators or inhibitors (e.g., odorant-binding proteins, odorant-binding proteins, other members of the lipocalin family, or members of the lipocalin family); G proteins; kinases (e.g., homologs of rhodopsin kinase and beta-adrenergic receptor kinase involved in receptor inactivation and desensitization); and compounds that alter the interaction between arrestin, which further inactivates and desensitizes the receptor, and the receptor. Modulators also include compounds that alter the affinity or transmission efficiency of ORs and alter the effect of activators on ORs. Modulators can include, e.g., genetically modified versions of OR family members with altered activity, as well as natural and synthetic ligands, antagonists, agonists, small chemical molecules, etc. Assays for such inhibitors and activators include, e.g., expressing an OR family member in cells or cell membranes and applying a putative modulator compound in the presence or absence of a flavor, fragrance, or malodor molecule, e.g., a malodor-causing substance such as DMTS, and measuring the functional effect on olfactory transmission as described above.A sample or assay containing an OR family member treated with a potential activator, inhibitor, or modulator is compared to a control sample that does not contain the inhibitor, activator, or modulator to examine the degree of regulation. A control sample (untreated with the modulator) is assigned a relative OR activity value of 100%. Inhibition of OR is achieved when the OR activity value relative to the control is about 80%, optionally 50%, or 25 - 0%.

[0027] As used herein, the terms "purified," "substantially purified," and "isolated" refer to a state in which the compounds of the invention are usually free of other different compounds that are associated with them in their natural state. "Purified," "substantially purified," and "isolated" subjects contain at least 0.5%, 1%, 5%, 10%, or 20% by weight, most preferably at least 50% or 75% by weight, of the mass of a given sample. In one preferred embodiment, these terms refer to a compound of the invention that contains at least 95% by weight of the mass of a given sample. As used herein, when referring to a nucleic acid or protein, the terms "purified," "substantially purified," "isolated," and "(substantially) isolated" also refer to a state of purification or concentration that is different from that which occurs naturally in mammals, particularly in the human body. A degree of purification or concentration that is higher than that which occurs naturally in mammals, particularly in the human body, including (1) purification from other related structures or compounds, or (2) association with structures or compounds that are not normally associated in the body of a mammal, particularly a human, enters into the meaning of "isolated." The nucleic acids or proteins or classes of nucleic acids or proteins described herein can be isolated or, if not, can be associated with structures or compounds that are not normally associated in nature according to various methods and processes known to those of skill in the art.

[0028] As used herein, the terms "amplify" and "amplification" refer to the use of any suitable amplification method to generate or detect recombinant forms of naturally expressed nucleic acids, as described in detail below. For example, the invention provides methods (e.g., by polymerase chain reaction, PCR) and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo dT primers) for amplifying naturally expressed (e.g., genomic DNA or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention in vivo, ex vivo or in vitro.

[0029] The term "seven-transmembrane receptor" refers to a polypeptide that belongs to a superfamily of transmembrane proteins that have seven domains that span the plasma membrane seven times (hence the seven domains are called "transmembrane" or "TM" domains, TM I-TM VII). The families of olfactory and certain taste receptors each belong to this superfamily. Seven-transmembrane receptor polypeptides have similar characteristic primary, secondary, and tertiary structures, as described in more detail below.

[0030] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single-stranded or double-stranded form. The term includes nucleic acids, i.e., oligonucleotides, that contain known analogs of natural nucleotides. The term also includes nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly depicted. Specifically, degenerate codon substitutions can be achieved, for example, by generating sequences in which the third position of one or more selected codons is replaced with mixed bases and / or deoxyinosine residues.

[0031] In addition to the gene sequences shown in the sequences disclosed herein, it will be apparent to those skilled in the art that variants also include DNA sequence polymorphisms that may exist within a given population that can lead to changes in the amino acid sequences of the polypeptides disclosed herein. Such genetic polymorphisms can be present in cells of different populations or within a population due to natural allelic variations. Allelic variants can also include functional equivalents.

[0032] Further embodiments relate to molecules derived from such sequence polymorphisms from specifically disclosed nucleic acids. These natural mutations typically result in about a 1 - 5% variation in the nucleotide sequence of the gene or the amino acid sequence of the polypeptides disclosed herein. As described above, the nucleic acids encoding the polypeptides of the embodiments herein are useful tools for modifying non - human host organisms or host cells intended for use in the methods described herein.

[0033] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, whether or not it includes natural and / or non - natural amino acids or polymers. The term "heterologous" when used in reference to a portion of a nucleic acid indicates that the nucleic acid contains two or more subsequences that are not found in the same relationship to each other in nature. For example, a nucleic acid is typically produced recombinantly and has two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein contains two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0034] "Promoter" is defined as the sequence of a nucleic acid sequence that directs the transcription of nucleic acids. As used herein, a promoter, in the case of a polymerase II type promoter, includes the necessary nucleic acid sequences near the transcription start site, such as the TATA element. Promoters optionally also include distal enhancer or repressor elements that can be thousands of base pairs away from the transcription start site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, or a sequence of a transcription factor binding site) and a second nucleic acid sequence, where the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0035] As used herein, "recombinant" refers to a polynucleotide (e.g., a "recombinant polynucleotide") that is synthesized in vitro or otherwise manipulated, a method of producing a gene product in a cell or other biological system using the recombinant polynucleotide, or a polypeptide (a "recombinant protein") encoded by the recombinant polynucleotide. Also, "recombinant" means, for example, the expression of a fusion protein, including a nucleic acid sequence potentially amplified using the transposition domain and primers of the present invention, such as the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for inducible or constitutive expression. "Recombinant" further means the modification of a cell resulting in the stable or transient expression of an endogenous gene, such as the receptor genes referred to herein, obtained by genome editing techniques such as CRISPR / Cas9.

[0036] The term "expression vector" refers to a recombinant expression system for constitutive or inducible expression of the nucleic acid sequences of the present invention in any cell, such as prokaryotic, yeast, fungal, plant, insect or mammalian cells, in vitro, ex vivo, or in vivo. This term includes linear or circular expression systems including, but not limited to, viral vectors, bacteriophages and plasmids. One skilled in the art can select an appropriate vector according to the expression system. This term includes expression systems that remain episomal or are integrated into the host cell genome. The expression system may or may not self-replicate, i.e., have the ability to promote transient expression in the cell. This term includes recombinant "expression cassettes" that may contain the minimal elements necessary for transcription of the recombinant nucleic acid. This term also covers, for example, cassettes or vectors for expression of endogenous genes via genome editing methods such as CRISPR / Cas9.

[0037] "Non-human organism or host cell" means a non-human organism or cell that contains the nucleic acid or expression vector described herein and supports the replication or expression of the expression vector. The host cell can be a prokaryotic cell such as E. coli, or a eukaryotic cell such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, HEK-293, for example, cultured cells, explants, and in vivo cells.

[0038] The term "tag" or "combination of tags" means a short polypeptide sequence that can be added to an odorant receptor protein. Usually, when DNA encoding a "tag" or "combination of tags" is added to DNA encoding a receptor, a fusion protein is ultimately obtained in which the "tag" or "combination of tags" is fused to the N-terminus or C-terminus of the receptor. Lucy, FLAG® and / or Rho tags can enhance receptor transport to the cell membrane and thus can assist in the expression of functional odorant receptors for in vitro cell-based assays [Shepard, B. et al., PLoS One 8, e68758–e68758 (2013), and Zhuang, H. & Matsunami, H. J. Biol. Chem. 282, 15284–15293 (2007)].

[0039] In one embodiment of the present specification, there is provided an isolated nucleic acid molecule comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, or its reverse complement.

[0040] In one embodiment of the present specification, there is provided the above-described isolated nucleic acid sequence encoding a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40.

[0041] In a further embodiment of the present specification, an isolated polypeptide is provided that comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40.

[0042] In one embodiment, a non-human organism or host cell is transformed to express a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40.

[0043] In one embodiment, a non-human organism or host cell is transformed to express a polypeptide comprising the same amino acid sequence as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40.

[0044] Furthermore, the present specification provides an expression vector comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40.

[0045] In addition, the present specification also provides an expression vector comprising a nucleic acid comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, or its reverse complement.

[0046] In addition, the present specification also provides an expression vector having a nucleic acid comprising a nucleotide sequence identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, or its reverse complement.

[0047] In one embodiment of the present specification, a method for identifying a compound that binds to, inhibits, blocks, inhibits, and / or regulates the activity of an olfactory receptor activated by a malodorous substance such as dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS), wherein the receptor is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40 and has an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, the method comprising: a) contacting the receptor or chimeric or fragment thereof with a compound; b) determining whether the compound affects the activity of the receptor is provided.

[0048] In one embodiment, a method for identifying a compound that binds to, inhibits, blocks, inhibits, and / or regulates the activity of an olfactory receptor activated by a malodorous substance, comprising: a. contacting a test substance and a malodorous substance with at least one olfactory receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1; b. measuring the response of the olfactory receptor in the presence and absence of the test substance to measure the response of the olfactory receptor to the malodorous substance; c. identifying a test substance that modulates the response of the olfactory receptor based on the responses measured in the presence and absence of the test substance; and d. Selecting the identified test substance as a compound that modulates the response of an olfactory receptor to an odor-causing substance comprising A method is provided herein, wherein the odor-causing substance is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

[0049] In a further embodiment of the present specification, a method for identifying an odor modulator, comprising a. Contacting a test substance and an odor-causing substance with at least one olfactory receptor comprising a polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40; b. Measuring the response of the olfactory receptor polypeptide to the odor-causing substance; c. Identifying a test substance capable of modulating or suppressing the response of the olfactory receptor based on the measured response; and d. Selecting a test substance that binds to, suppresses, blocks, inhibits, and / or modulates the response of the olfactory receptor as an odor modulator comprising A method is provided herein, wherein the odor-causing substance is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

[0050] One embodiment is a method for identifying an odor suppressant, comprising a. Contacting at least one olfactory receptor with a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40, and a substance causing malodor; b. Measuring the response of the olfactory receptor polypeptide to a substance causing malodor; c. Identifying a test substance capable of suppressing the response of the olfactory receptor based on the measured response; and d. Selecting the test substance that suppresses the response of the olfactory receptor as a malodor suppressant comprising wherein the substance causing malodor is DMTS and the malodor is fecal odor or halitosis, said method.

[0051] One embodiment is a method for identifying a compound that putatively modulates DMTS-related malodor, comprising: (i) contacting a cell line expressing a DMTS receptor polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40 with at least one compound; (ii) screening for a compound that binds to, suppresses, blocks, inhibits, and / or modulates the activity of the olfactory receptor polypeptide; and (iii) identifying a compound that putatively modulates DMTS-related malodor if it binds to, suppresses, blocks, inhibits, and / or modulates the activity of the DMTS receptor polypeptide, said method.

[0052] In a further embodiment, the malodor-causing substance in the method described herein is dimethyl trisulfide (DMTS).

[0053] Furthermore, a. a nucleic acid comprising a combination of tags including one or more of Lucy, FLAG (registered trademark), and / or Rho tag; and b. a nucleic acid encoding a receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR4S2, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1 or its complement A recombinant nucleic acid molecule is provided that comprises the same.

[0054] In a further embodiment, the Lucy tag comprises SEQ ID NO: 47, the FLAG (registered trademark) tag comprises SEQ ID NO: 43, and the Rho tag comprises SEQ ID NO: 45.

[0055] Furthermore, there is provided any one of a plurality of malodor-regulating compounds that bind, suppress, block, inhibit, and / or modulate the activity of an olfactory receptor activated by a malodor-causing substance such as dimethyl trisulfide (DMTS) and identified by the methods disclosed herein.

[0056] In one embodiment herein, there is provided a malodor-regulating compound that binds, suppresses, blocks, inhibits, and / or modulates the activity of at least one olfactory receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1 and is identified by the methods disclosed herein.

[0057] Another embodiment relates to the use of a polypeptide that is activated or activatable by DMTS or DMDS and comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40 for identifying a malodor modulating compound that binds to, inhibits, blocks, inhibits, and / or regulates the activity of an olfactory receptor.

[0058] Furthermore, there is provided a cell recombinantly modified to express the above polypeptide.

[0059] In one embodiment herein, there is provided a non-human host organism or host cell transformed or modified to express a receptor activated by DMTS selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR4S2, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1.

[0060] In one embodiment herein, there is provided a non-human host organism or host cell transformed or modified to express a receptor activated by DMTS, wherein the receptor comprises a polypeptide encoded by a polypeptide described herein or a nucleic acid described herein.

[0061] In a further embodiment herein, there is provided a non-human host organism or host cell comprising a nucleic acid or expression vector described herein.

[0062] In one embodiment, the cells provided herein are prokaryotic cells. In another embodiment, the cells provided herein are eukaryotic cells. In certain embodiments, the cells provided herein are selected from the group consisting of yeast cells and plant cells. In more specific embodiments, the cells provided herein are selected from the group consisting of HEK293, CHO, Xenopus oocytes, COS, yeast, bacteria, and cells derived from the olfactory placode.

[0063] To identify unknown DMTS or DMDS-specific receptors, dissociated olfactory sensory neurons (OSNs) can be screened using DMTS and DMDS. DMTS and DMDS can be further used in cell-based dose-response experiments performed on specific DMTS or DMDS receptors to evaluate both receptor specificity and sensitivity.

[0064] In one aspect of the invention, there is provided a method for identifying mammalian odorant receptors for malodor regulating compounds, and the use of the receptors for screening, particularly high-throughput screening (HTS) of malodor regulators (e.g., those that bind, inhibit, block, suppress, and / or modulate the activity of ORs).

[0065] In particular, herein, mouse receptors such as Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr669, Olfr665, Olfr207, Olfr1211, and their human counterparts OR4S2, OR5T1, OR8H3, OR2L13, OR2AJ1, OR11G2, OR52N5, OR52N2, OR5AC2, and OR4C15 are provided as receptors for the malodor-causing substances DMTS, DMDS, or other polysulfide compounds as shown in Table 1.

Table 1

[0066] While not wishing to be bound by theory, the mouse receptor Olfr738 is a paralog of the receptor Olfr742, and the human OR11G2 is an ortholog of these mouse receptors; and the mouse receptor Olfr669 is a paralog of the receptor Olfr665 and an ortholog of the human receptor OR52N5; and the mouse receptor Olfr665 is an ortholog of the human receptor OR52N2. Some receptors have previously been identified in WO 2014 / 210585 as indole and / or skatole-responsive odorant receptors. However, these receptors have not previously been associated with the malodor-causing substances DMTS or DMDS or other related polysulfide compounds. Since indole and skatole are not chemically related to polysulfide compounds, DMTS would not be defined as a potential agonist of these receptors by a chemical structure-based approach such as structure-activity relationship (SAR) to identify additional ligands.

[0067] In a further embodiment, the indicator for monitoring the activity of an olfactory receptor is selected from a fluorescent calcium indicator dye, a calcium indicator protein, a fluorescent cAMP indicator, a cell mobilization assay, a cell dynamic mass redistribution assay, a label-free cell-based assay, a cAMP response element (CRE)-mediated reporter protein, a biochemical cAMP HTRF assay, a beta-arrestin assay, or an electrophysiological recording. In particular, a calcium indicator dye is selected, which can be used to monitor the activity of olfactory receptors expressed on the membrane of olfactory neurons (e.g., Fura-2 AM).

[0068] In certain embodiments, compounds are screened continuously and odorant-dependent changes in calcium dye fluorescence are measured using a fluorescence microscope or a fluorescence-activated cell sorter (FACS).

[0069] In a further embodiment, molecular 3D receptor modeling of olfactory receptors is used to evaluate binding ability in silico and identify compounds that activate, mimic, block, inhibit, modulate, and / or enhance the activity of olfactory receptors.

[0070] As an example, olfactory neurons activated by DMTS or DMDS are isolated using a glass microelectrode or FACS machine attached to a micromanipulator. Mouse olfactory neurons are Ca imaged in a manner similar to the previously described procedure [Malnic, B. et al. Cell 96, 713 - 723 (1999); Araneda, R. C. et al. J. Physiol. 555, 743 - 756 (2004); and International Publication No. WO 2014 / 210585, which is hereby incorporated by reference in its entirety]. In particular, a motorized microscope stage is used to increase the number of cells that can be screened to at least 1,500 per experiment. Since mice have approximately 1,200 different olfactory receptors and each olfactory neuron expresses only one of the 1,200 olfactory receptor genes, this screening ability will substantially cover the entire mouse odorant receptor repertoire. In other words, the combination of calcium imaging for high - throughput olfactory neuron screening leads to the identification of almost all odorant receptors that respond to a particular profile of odorants. In certain embodiments, odorant receptors that respond to DMTS or DMDS can be isolated. For example, at least one neuron is isolated. 2+ Screened by imaging [Malnic, B. et al. Cell 96, 713 - 723 (1999); Araneda, R. C. et al. J. Physiol. 555, 743 - 756 (2004); and International Publication No. WO 2014 / 210585, which is hereby incorporated by reference in its entirety]. In particular, a motorized microscope stage is used to increase the number of cells that can be screened to at least 1,500 per experiment. Since mice have approximately 1,200 different olfactory receptors and each olfactory neuron expresses only one of the 1,200 olfactory receptor genes, this screening ability will substantially cover the entire mouse odorant receptor repertoire. In other words, the combination of calcium imaging for high - throughput olfactory neuron screening leads to the identification of almost all odorant receptors that respond to a particular profile of odorants. In certain embodiments, odorant receptors that respond to DMTS or DMDS can be isolated. For example, at least one neuron is isolated.

[0071] In calcium imaging of olfactory neurons, the main olfactory epithelium can be dissected from the mouse before the neurons dissociate. The dissected olfactory epithelium can be transferred to dissociation buffer for mechanical and enzymatic dissociation. The dissociated neurons are then seeded onto coverslips to enable screening of thousands of cells by fluorescence microscopy, and a calcium-sensitive dye (Fura-2 AM) is loaded into the cells, for example, at 31 °C for about 30 minutes, and the cells can be transferred to a microscope ready for screening. The cells are stimulated by perfusing a dilute solution of odorant (in saline) over the dissociated olfactory neurons. Rare cells that respond to malodorous compounds stimulate the receptor with, for example, a 50 μm malodorous compound and then the intracellular Ca 2+ flux is identified by monitoring the change in Fura-2 fluorescence. After analysis, responding cells can be recovered from the glass coverslip with a suction micropipette. The isolated cells are then either pooled into one sample or processed individually for subsequent identification of the odorant receptor genes expressed as mRNA in the responding cells.

[0072] In certain embodiments, the mRNA of olfactory neurons is purified and amplified according to the methods generally described in Marko, N. F. et al., (2005) A robust method for the amplification of RNA in the sense orientation. BMC genomics, 6, 27; doi:10. 1186 / 1471-2164-6-27 (Eberwine method). At least a portion of the transcriptome (including the entire transcriptome) is sequenced using next-generation sequencing (NGS) techniques or hybridized to known genes using microarray techniques. NGS is generally described and is described in Metzker, M. L. Nat. Rev. Genet. 11, 31-46 (2010). In certain embodiments, at least five neurons showing the same response profile are pooled. The mRNA is released by cell lysis immediately after collection; the DNAse and purification steps are not performed. The mRNA is amplified by two consecutive in vitro transcriptions (IVT). The amplification can be performed with the following parameters according to the MessageAmpII aRNA kit (Ambion, AMA1751): two consecutive 14-hour IVTs.

[0073] In a further embodiment, the mRNA of a single olfactory neuron is purified and amplified by an LD-PCR (long-distance polymerase chain reaction)-based method such as that described in an NGS-ready kit (e.g., Clontech / Takara, SMARTer® Ultra® Low Input RNA Kit for Sequencing-v3, cat.634848). The mRNA of a single cell is first reverse transcribed into the corresponding cDNA and subsequently amplified in 18 PCR cycles to function as an NGS sample for transcriptome sequencing.

[0074] In yet another embodiment, the identity of a group of DMTS olfactory receptors or gene family is determined by comparing the results of NGS reads obtained from isolated activated olfactory neurons to a reference genomic sequence of the same species (e.g., up to the number of selected neurons). In particular, the putative DMTS receptor will be the most abundant olfactory receptor mRNA in an NGS sample from olfactory neurons or will be present in multiple independent biological replicates. Due to the combinatorial nature of the olfactory code (one compound can activate many ORs and one OR can be activated by many compounds), pooling several neurons activated by a particular compound enables the search for substantially all receptors involved in the perception of these molecules in a single NGS experiment. Thus, pooling functionally similar neurons significantly improves the orphaning throughput and speed.

[0075] Subsequently, using standard bioinformatics tools, under the assumption that homologous sequence receptors retain similar functions, human odorant receptors most closely related to other putative mammalian (non-human) DMTS receptors are identified. Several methods have successfully identified human OR-ligand pairs based on this assumption [Armelin-Correa and Malnic (2017)], and it is thought that up to 80% of mouse-human orthologs maintain similar functional response profiles [Adipietro, K.A et al., PLoS Genet. 8, e1002821-e1002821 (2012)]. The BLASTP and / or BLASTN algorithms, or other ortholog pair identification algorithms, such as the default parameters of InParanoid, can be used.

[0076] The DMTS receptors of humans or non-human mammals can be adapted to functional assays that can be used to identify compounds that bind to, inhibit, block, suppress, and / or modulate the activity of olfactory receptors. In particular, the assay can be a cell-based assay or a binding assay, and the method for identifying the compound can be a high-throughput screening assay. More specifically, provided herein are receptor-based assays that are adaptable for high-throughput screening of receptors using compound libraries for the discovery of modulatory compounds (e.g., binding, blocking, inhibiting, suppressing, and masking).

[0077] In certain embodiments, the DMTS receptor gene sequences are identified from DMTS-sensitive cells as follows: Pooled neurons are heated to 75° C. for 10 minutes to disrupt the cell membrane and make their mRNA available for amplification. This amplification step is important when applying NGS technology to limited amounts of starting material (usually 1-15 cells). Linear amplification by the Eberwine method (IVT) ensures the maintenance of the relative transcription levels of the expressed genes. Using in vitro transcription for two consecutive nights (14 hours), a sufficient amount of cRNA is obtained; the amplified cRNA is then used to generate an Illumina HiSeq cDNA library. The resulting short sequences (generally referred to as “reads”), usually 75-150 base pairs, are aligned against the mouse reference genome (e.g., UCSC version mm9 or mm10) to construct the complete transcriptome of these cells. Quantitative analysis of the transcriptome data yields a list of the transcribed odorant receptor genes and their respective expression levels. Odorant receptor genes that exhibit the most abundant levels of mRNA (the most abundant “reads”) or are present in multiple replicate experiments are considered putative DMTS receptors.

[0078] Next, using the predicted mouse OR genes, the most recent versions of genomic databases for both mouse and human are mined to identify the most closely related receptors in mouse (paralogous genes) and human (orthologous genes), i.e., those with the highest sequence similarity. This process can be carried out using the BLAST search algorithm, a sequence similarity search tool (published on the NCBI website), where all the putative gene sequences previously obtained in the initial transcriptome analysis are used as query sequences. The newly identified genes identified from this data mining process are considered potential DMTS receptors under the assumption that paralogous and orthologous genes are likely to have similar activities. In certain embodiments, a pairwise sequence homology comparison is performed to identify closely related receptors in mouse and human, and the receptors are identified as described in International Publication No. WO 2014 / 210585. Other approaches such as RT-PCR and microarray approaches may also be used.

[0079] In a further embodiment, to complete the deorphanization process, candidate OR genes are further expressed in vitro and their activity against compounds used to isolate olfactory neurons and other structurally related target compounds is confirmed. Mouse receptors identified from isolated olfactory neurons responsive to DMTS are modified at their N-termini with short polypeptide sequences (e.g., FLAG® (SEQ ID NO: 44), Rho (SEQ ID NO: 46; first 20 amino acids of the bovine rhodopsin receptor), and / or Lucy (SEQ ID NO: 48; cleavable leucine-rich signal peptide sequence) tags), transiently expressed in HEK 293T cells, and individually stimulated with DMTS to confirm their identity as bona fide DMTS receptors. In a further embodiment, the RTP1 gene can also be expressed in cell lines, whether by activation of the endogenous RTP1 gene or by transformation. The human G alpha subunit Gα in this cell-based assay olfCo-expression activates the Gs signaling pathway that leads to an increase in intracellular cAMP upon binding to the appropriate ligand. Alternatively, co-expression of human G alpha subunit Gα 15 activates the Gq signaling pathway that leads to an increase in intracellular Ca 2+ upon binding to the appropriate ligand. The above processes and the results obtained so far help to validate the process of rapidly and reliably identifying mammalian odorant receptors for DMTS or DMDS.

[0080] Furthermore, an assay for identifying compounds that bind to the odorant receptor for DMTS or DMDS is provided. In a further embodiment of the present specification, at least one olfactory receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, OR52N5, OR2L13, OR4C15, OR5AC2, OR8H3, OR11G2, and OR52N2 is provided, which binds, inhibits, blocks, inhibits, and / or regulates the activity of the olfactory receptor, and an odor-modulating compound identified by the method described herein, for example, the compound described in FIG. 4.

[0081] In one embodiment, the activity of the compound is determined by comparing its binding to the binding of DMTS or DMDS. In another embodiment, the receptor or chimeric or fragment thereof is contacted with the compound in the presence of DMTS or DMDS under conditions that allow the compound to bind to the receptor together with DMTS or DMDS.

[0082] In a further embodiment, the compound is contacted with a receptor or chimeric or fragment thereof that is activated by DMTS or DMDS, wherein the receptor or chimeric or fragment thereof is expressed in a cell that has been recombinantly modified to express the receptor polypeptide.

[0083] The activity of the compound can be determined using in vivo, ex vivo, in vitro and synthetic screening systems.

[0084] In another embodiment, the contact is carried out with liposomes or virus-induced budding membranes containing the polypeptides described herein.

[0085] In another embodiment, the method for identifying a compound that binds, inhibits, blocks, inhibits, and / or modulates the activity of an olfactory receptor that can be activated by DMTS or DMDS can be carried out in intact cells expressing the polypeptides described herein or in membrane fractions from cells, or in cultured olfactory neurons modified to express endogenous or exogenous odorant receptors.

[0086] Accordingly, the 21 ORs described herein constitute valuable candidate receptors for the identification of modulators, antagonists, and / or blockers that are involved in the perception of malodors induced by DMTS or DMDS and that modulate, reduce, suppress, inhibit, and / or block the perception of malodors.

[0087] The nucleic acid and amino acid sequences identified and / or used herein are listed below: Olfr1193 DNA-sequence number 1 atggcctcaaggacttattccatggaagaagtaaataatgtcactgaattcattttcttgggtctttctcagaaccctgaggttgaaaaagtgtgctttgtggtgttctccttcttttacatggtcattctgctaggaaacctcctcatcatgttgacagtttgcagtggcaatcttttcaagtttcccatgtattttttcctcaactttctgtcttttgtggacatttgctactcctcagtcacagcacccaagatgattattgacctgttagtgaagaaaaagactatatcctatgtggggtgcatgttacaactctttgtggttcatttctttggttgcactgagatcttcattcttactgtcatggcctatgatagatatgtggccatttgtaaacctctccactatatgactatgatggaccgggaaagatgcaataagatgttgctcggaacatggatcggtggcttcttacattctattatccaagtggctcttgtggtccagctccccttttgtggaccgaatgagattgatcactatttctgtgatgtacatcctgtactgaaacttgcctgcactgacacttacattgttggtatttttgtgacagcaaacagtggcaccattgcattgggaagttttgtcatcttgctgatctcatacacagtcattctcatgtctctgagaaagcagtcatctgaaggcagacgcaaagctctctccacttgtggatcccacattgctgttgtcatcattttttttggcccctgtacttttatgtatatgcggcctgacactaccttctctgaggacaagatggtagctatattttacaccattatcactcccatgctgaatcctctaatttacactctaagaaatgcagaagtaaagaatgcaatgagaaaactgtgggctagaaagttttcctgggaaactactgggaaatag Protein - SEQ ID NO: 2 MASRTYSMEEVNNVTEFIFLGLSQNPEVEKVCFVVFSFFYMVILLGNLLIMLTVCSGNLFKFPMYFFLNFLSFVDICYSSVTAPKMIIDLLVKKKTISYVGCMLQLFVVHFFGCTEIFILTVMAYDRYVAICKPLHYMTMMDRERCNKMLLGTWIGGFLHSIIQVALVVQLPFCGPNEIDHYFCDVHPVLKLACTDTYIVGIFVTANSGTIALGSFVILLISYTVILMSLRKQSSEGRRKALSTCGSHIAVVIIFFGPCTFMYMRPDTTFSEDKMVAIFYTIITPMLNPLIYTLRNAEVKNAMRKLWARKFSWETTGK Olfr1093 DNA sequence number 3 atggaaaagatcacatcagctgtggatgtccacaatattccattaaagaacatgactgaagccaccatgtttattctcttaggattcacagatgactttgaactccaagtcttcctgtttttactgtttcttgctatttatctcttcactctggtaggaaactttggactggttgttttggtcattggggattgtcggctacacaaccccatgtactatttcctaagtgttttgtctttcctggatgcttgctattctacagttgttacacccaaaatgttggtcaactttctaagtgaaaataagtccatttcattccttgcatgtgcaacccaaatgcttctctttgtttcgttgggaaccacagaatgctttctcctggcagcaatggcttatgaccgatatgtagccatctacaacccacttctgtatacagtggccatgtcacccagagtatacctgccactcatcattgcttcctatgctggtggagttgtgcatggtgctatccacacagtggccactttcagtctgtccttctgtggatccaatgaaattaagcatgtcttctgtgacatccctgcattgcttgctctttcttgttctgatacccacacaaatgagcttctagtcttgtacttggtgggcttgattgagattgttaccatcctgattgttctggtctcctatggattcatcctctttgccattctgaacatgcattctgctgagggtaggaggaaagtgttctctacatgtggctctcacctcactggagtctctatttaccatggtacaatccttttcacttatatgaggcctagttccagttatgcttcaaatcatgacatggtagtgtcaatattttacaccattgtgatacccatgttgaatcctatcatctatagtttgaggaacaaagatgtaaaagtagcatttaataaattgtggagaaaatgtgattcataa Protein - SEQ ID NO: 4 MEKITSAVDVHNIPLKNMTEATMFILLGFTDDFELQVFLFLLFLAIYLFTLVGNFGLVVLVIGDCRLHNPMYYFLSVLSFLDACYSTVVTPKMLVNFLSENKSISFLACATQMLLFVSLGTTECFLLAAMAYDRYVAIYNPLLYTVAMSPRVYLPLIIASYAGGVVHGAIHTVATFSLSFCGSNEIKHVFCDIPALLALSCSDTHTNELLVLYLVGLIEIVTILIVLVSYGFILFAILNMHSAEGRRKVFSTCGSHLTGVSIYHGTILFTYMRPSSSYASNHDMVVSIFYTIVIPMLNPIIYSLRNKDVKVAFNKLWRKCDS Olfr1097 DNA sequence number 5 atgagtgcctgtaatcatacaaatgaacctgagttcacgcttgtgggactgacagactccaaggagattcagctggtcctctctgttttgtttctcctgatatacatgctcactgtcttgggaaacataggtatgatactgatcattcatctagatgtccagctccacactccaatgtattttttcctcacccacttgtcattccttgacctcagttactcaactgtaatcacacctaaaaccttacagaatacgctgacctccataaaaaatatttccttcatgggatgcttcacccagttgtatttctttgtcctcttggcagcttctgaatgttttatactttcgtcaatggcctatgaccgctatgtagctatctgcaaccctctacactatccagttattatgtcccctaggcgctcatatactctcatcactgtgtcctacatgattggagttttggattcttctgtcactgtcttttgcttaagcacactggatttctgcaactccaaagtaattcatcacttcttttgtgacacattcccaattttagctctgtcctgcagtgatacctataatgcagaagccactatattcgttttagctggttccactctattgctgtcgctcatcacgatatcctcatcctatgtatctattctctctacaattttgaagataaattcttcttcaggaaagcacaaagccttctctacatgtgcctcacatcttataggagtcactgttttttatggtacaatgatctttacttatttaaaaccaagtacgtcctactccctgggaaaggatcaagtagcctctgttttttatactatagtgattcccatgctgaacccacttatctatagtctcaggaacaaagaagtgaaaagtgctgttgttagagttatgaagaagagagagtgcatccagaaactagaataa Protein - SEQ ID NO: 6 MSACNHTNEPEFTLVGLTDSKEIQLVLSVLFLLIYMLTVLGNIGMILIIHLDVQLHTPMYFFLTHLSFLDLSYSTVITPKTLQNTLTSIKNISFMGCFTQLYFFVLLAASECFILSSMAYDRYVAICNPLHYPVIMSPRRSYTLITVSYMIGVLDSSVTVFCLSTLDFCNSKVIHHFFCDTFPILALSCSDTYNAEATIFVLAGSTLLLSLITISSSYVSILSTILKINSSSGKHKAFSTCASHLIGVTVFYGTMIFTYLKPSTSYSLGKDQVASVFYTIVIPMLNPLIYSLRNKEVKSAVVRVMKKRECIQKLE Olfr166 DNA Sequence No. 7 atggagaaatggaatcagagctcaagtgattttactctgttaggactgcttccacaaaaccaaacaggcctgctacttttgatgctcatcatctttgtcttctctctggctttgtgtggcaactcaggaatgatccacctcattcgtgtggatccaaggctccacacccccatgtactttctcctcagtcagctctctctcatggacctgatgtacatttctaccactgttcccaagatggcatttaacttcctttctggccagaaaagcatctcttttctgggctgtggagtgcaatccttcttcttcctgactatggcatgttctgagggcttgctcttggcttccatggcttatgatcgttttgtggctatctgccatccccttcactatcccattcgcatgagcaaaataatgtgtctgaagatgatcataggatcctggatattgggctcaatcaactctttagcacataccgtctatgcccttcatattccttactgccattctaggtccattaaccatttcttctgtgatgttccagccatgttgcccctggcctgtatggacacttgggtttatgagtacatggtgtttgtgagcacaagcctgtttctcctactgcctttccttggtatcacagcttcctatggtcgggtcctttttgctgtcttccacatgcgctcaaaagagggaaagaagaaggccttcaccacatgctcaactcacttaactgtggtgacattttactatgcaccttttgtctatacctatcttcgacctaggagtcttcgctccccaacagaagataagattctggctgttttctacactatccttacccccatgctcaaccccatcatttatagtctgaggaataaggaggtcctgggggccatgacaagagtccttggtacttttccttcaactaaaccgtaa Protein - SEQ ID NO: 8 MEKWNQSSSDFTLLGLLPQNQTGLLLLMLIIFVFSLALCGNSGMIHLIRVDPRLHTPMYFLLSQLSLMDLMYISTTVPKMAFNFLSGQKSISFLGCGVQSFFFLTMACSEGLLLASMAYDRFVAICHPLHYPIRMSKIMCLKMIIGSWILGSINSLAHTVYALHIPYCHSRSINHFFCDVPAMLPLACMDTWVYEYMVFVSTSLFLLLPFLGITASYGRVLFAVFHMRSKEGKKKAFTTCSTHLTVVTFYYAPFVYTYLRPRSLRSPTEDKILAVFYTILTPMLNPIIYSLRNKEVLGAMTRVLGTFPSTKP Olfr169 DNA - Sequence number 9 atggaatatgagaactacacttttaacagcgacttcatcctcttgggactgttctcttcttcaaagacaagcttaacttttttctcatttatatttttcatttttattatggctataacagaaaatgccctcatgatcctcctaatccacagggattctcgactccataccccaatgtatttcctgcttagtcatctctccttcatggatatcttgcacatttccaacattgttcctaaaatgattgctgacttcctctcaggcagcagaactatttcctttgcaggctgtgccttccagatatttctctctcttaccttgctaggtggtgagtgccttctcctggcagccatgtcctatgatcgatatgtggccatctgccacccacttcgctaccctgtgctgatgagggataactccagtaggctcctggctgcaggctcctggctggtggggatcctcaactccatagtacacacagtttttgcactccactttcccttctgccactcaagagccattgatcactttttctgcgaagtccctgccatgttgaaattgtcatgtatagacacaacacactatgaacgaggcgtttatgtgagtggcattatttttctgctgatcccattttccatgatctctatatcttatgtgcaaattctcctcactgtattccaaatgcagtcatcaggggcccggcaaaagtccttttccacctgttccttccacatggttgttgtcataatgtactatgggccattcatttttacatatatgagacctcgctcataccacactccagggcaggataaatttttggcaatattctacaccatcctgacacccacactcaaccccataatctacagctttcgtaataaagatgtccttatggctgtgaaaaacatcgtccaaagtaattttttgaataaaaaatga Protein - SEQ ID NO: 10 MEYENYTFNSDFILLGLFSSSKTSLTFFSFIFFIFIMAITENALMILLIHRDSRLHTPMYFLLSHLSFMDILHISNIVPKMIADFLSGSRTISFAGCAFQIFLSLTLLGGECLLLAAMSYDRYVAICHPLRYPVLMRDNSSRLLAAGSWLVGILNSIVHTVFALHFPFCHSRAIDHFFCEVPAMLKLSCIDTTHYERGVYVSGIIFLLIPFSMISISYVQILLTVFQMQSSGARQKSFSTCSFHMVVVIMYYGPFIFTYMRPRSYHTPGQDKFLAIFYTILTPTLNPIIYSFRNKDVLMAVKNIVQSNFLNKK Olfr738 DNA Sequence No. 11 atgaaagcctttagcagccccagcaactccagcatcatcactggcttcatcctcctgggcttcccctgccccaaggaggggcaaatcctcctctttgtgctcttcttcattatctacatccttaccctcatgggcaatgcttccatcatatgtgctgtgtgctatgataagaaacttcacagccccatgtacctcctgctggccaacttctccttcctagaaatctggtatgtcacctccacagtccccaacatgttggccaacttcctctctgacacgaaggtcatctctttctctggatgcttcctgcagttctatttcttcttctccttgggttctacagaatgctttttcctggcagtcatggcatttgatcgataccttgccatctgcagacctctacattatccttctctcatgactgggcgcctctgcaacatccttgtgatcagttgctgggtgcttggtttcctctggttccctgttcccatcatcatcatctcccaaatgtccttctgtggatccagaattatagaccacttcctgtgtgacccaggccctctgttggccctcacctgtgtgagaaattctttaattgagatgactagctctactttaagttccctgcttttatttgttccatttttttttatcatggggtcttatgctctagtaatgagggctgtgctcagggtcccttcagcagctggacgaagaaaggccttctccacctgtgggtcacacttgactgtggtttctcttttctatggctcagtgatggtcatgtatgtgagcccaacatctgaacatgcagctggagtgcaaaaacttgtgactctgttttattctgtggttactcccctccttaatcctgtgatatacagtctgaggaacagagatatgaaacatgcaatgaaaaagttactgaaaatgtaa MKKPFSSPQQLQHITGFIPPGFPLPKGGKPPLFVLFFIILTITPHWQAASIIVCCCDDKKLFTPHYLLANFLFLRNLGMSHSTQPMLANFLSDTKVISFLDAFPAFYFFFLGFYRNALFPASWIFDRTCAICRTLYIIFSMTGRLCNILVIQLGLGFLLVPVPPSPSQNVLSVDPRYRTHFCVTQALVAPTVEKLFIEDTLSTFKFPCFIFVPFFFFFFMGSLALSNEGCASSGPFSAGDEEGFSHTVGTLDVGFSSYGSVDGVYVSPNIEHAEGCKKLVTLCFYLVYSPPNPVITSETEYETANESSYEN Protein - SEQ ID NO: 12 MKAFSSPSNSSIITGFILLGFPCPKEGQILLFVLFFIIYILTLMGNASIICAVCYDKKLHSPMYLLLANFSFLEIWYVTSTVPNMLANFLSDTKVISFSGCFLQFYFFFSLGSTECFFLAVMAFDRYLAICRPLHYPSLMTGRLCNILVISCWVLGFLWFPVPIIIISQMSFCGSRIIDHFLCDPGPLLALTCVRNSLIEMTSSTLSSLLLFVPFFFIMGSYALVMRAVLRVPSAAGRRKAFSTCGSHLTVVSLFYGSVMVMYVSPTSEHAAGVQKLVTLFYSVVTPLLNPVIYSLRNRDMKHAMKKLLKM Olfr742 DNA Sequence No. 13 Atgaaaaccctcagcagccccagcaactccagcaccatcactggcttcatcctcttgggcttcccctgccccagggaggggcaaatcctcctctttgtgaccttcttcattgtttacatactcattcttatgggcaatgcttccatcatctgtgctgtgtactgtgatcagagcctccacacccccatgtacttcctgctggccaacttctccttcctggagatctggtatgtcacctccacagtccccaacatgttggccaacttcctttcagacaccaaggtcatctctttctctggatgcttcctgcagttctatttcttcttctcctttggttctacagaatgctttttcctggcagtcatggcatttgatcgataccttgccatctgtaggccactacattatccttctctcatgactgggcacctctgcaacatccttgtgatcagttgctgggtgcttggtttcctctggttccctgtacccatcatcatcatctcccagatgtccttctgtgggtccagaattatagaccacttcctgtgtgacccaggccctcttttggcccttgcctgttccagagccccattgatggaggttttctggacaattataatgtctatgctcctggttattcctttcctcttcatcatgggaacttacatattggtcctaagagctgtgtttagacttccttcaagagatggacaaaaaaaggccttctccacttgcgggtctcatctcacagtagtttcactcttttattgctcagtgatgaaaatgtatttgagcccaacatctgagcatgaagctggaatgcagaagcttgtaactctattttattctgtgggtactccactacttaatcctgtgatatacagtctgaggaacaaagatatgaaaaatgccctgcagaagattttaagaacataa Protein - SEQ ID NO: 14 MKTLSSPSNSSTITGFILLGFPCPREGQILLFVTFFIVYILILMGNASIICAVYCDQSLHTPMYFLLANFSFLEIWYVTSTVPNMLANFLSDTKVISFSGCFLQFYFFFSFGSTECFFLAVMAFDRYLAICRPLHYPSLMTGHLCNILVISCWVLGFLWFPVPIIIISQMSFCGSRIIDHFLCDPGPLLALACSRAPLMEVFWTIIMSMLLVIPFLFIMGTYILVLRAVFRLPSRDGQKKAFSTCGSHLTVVSLFYCSVMKMYLSPTSEHEAGMQKLVTLFYSVGTPLLNPVIYSLRNKDMKNALQKILRT Olfr207 DNA - Sequence No. 15 atggaactgaacaggacccagctgactgaatttgttctcagaggaataacagatcgttcagagctgcaagtccccctgttcctggtgttctttctcatctatgttatcaccatggtgggcaaccttggcttaatctttgtcatctggaaggaccctcatcttcacacacccatgtaccttttccttggaaatttggcctttgctgatgcctgtaattcatcctctgtgacaccaaagatgcttatgaaatttttaaataagaatgacatgatatccatgggtgagtgttttgctcaattttatttcttttgttcaagtgtaactgcagaagccttcattctggtagctatggcctatgaccgctatgtagccatatgcaaacctctgctctatgtagtggtgatgtccaacagactctgtattcagttcataggtgtatcctatctaattggacttctacatggcttacttcatgtaggattgttatttaggttaacgttttgtagttccaatgtaatagattatttctactgtgacatcctgccactttataggatttcttgcactgacccatcgatcaatgtactggtagctttcattatgggtattttattacaagtgagtacctttatgagtattatagtctcctatgtccgtgtcctctttgccatcctgagaacaaagtctgagaggggcagaaacaaagccttctctacttgcagttcccacctgtcatctgtgtctttgttctatggcactctcttcatcatatatgtcctctctggctctgacacagataattatcagggtaaaatgtattcactgttctataccattatcattcctctgctaaaccccttcatttacagcctaagaaataaagaagtcatcggtgccttgagaaaagtcagaaaatga Protein - SEQ ID NO: 16 MELNRTQLTEFVLRGITDRSELQVPLFLVFFLIYVITMVGNLGLIFVIWKDPHLHTPMYLFLGNLAFADACNSSSVTPKMLMKFLNKNDMISMGECFAQFYFFCSSVTAEAFILVAMAYDRYVAICKPLLYVVVMSNRLCIQFIGVSYLIGLLHGLLHVGLLFRLTFCSSNVIDYFYCDILPLYRISCTDPSINVLVAFIMGILLQVSTFMSIIVSYVRVLFAILRTKSERGRNKAFSTCSSHLSSVSLFYGTLFIIYVLSGSDTDNYQGKMYSLFYTIIIPLLNPFIYSLRNKEVIGALRKVRK Olfr665 DNA sequence number 17 atgcctggggtcaatacctccagcctgacaccaagatactttattctcaatgggattcctgggttggaagctgcacacatctggatctctctgccattcttcattatgtacctcattgctgtcacaggtaactgtggacttatctacctcatcagtcatgaggaggctctgcaccggcccatgtactactttctagccatgttgtctgctacagatatttctgggtgtaatacaattgtccccagtatgttatgcatcttttggttcagtgtcaaggagattgatttcaatgcctgccttgtacagatgtttttcatccacatgttaacaggcatggagtctggtgtgctcatgcttatggctctcgaccgctatgtggctatatgctatccattacgctatactaccatactcaccaacactatgattaccaagattggattggcagcacttgttagaagtgtgttactcatggtcccttttgctttcctgatcaagcgtcttccatactgtagaggaaacctcatccaacatacctattgtgatcacatggctgtggctaaactatcctgtggcaatattaagattaatgctatctatggtcttataattgctatatttattgggggttttgatatattctgtatctccatgtcttatgccatgattatccatgctgtggtgaagctatcttcggcagatgctcgccataaagccttcagtacctgtacatcacacatatgtgctattgttattacctatgtcccagcattcttcaacttctttactcatcgctttgggagaaccactataccccatcatatccacattattatagccaacctgtatctattgctacctcccaccttgaatccaattgtatatggagtaaagaccaagcagattcgtgaaggtgtgatcaaactgtttgctagacaaaaagttgtttga Protein - SEQ ID NO: 18 MPGVNTSSLTPRYFILNGIPGLEAAHIWISLPFFIMYLIAVTGNCGLIYLISHEEALHRPMYYFLAMLSATDISGCNTIVPSMLCIFWFSVKEIDFNACLVQMFFIHMLTGMESGVLMLMALDRYVAICYPLRYTTILTNTMITKIGLAALVRSVLLMVPFAFLIKRLPYCRGNLIQHTYCDHMAVAKLSCGNIKINAIYGLIIAIFIGGFDIFCISMSYAMIIHAVVKLSSADARHKAFSTCTSHICAIVITYVPAFFNFFTHRFGRTTIPHHIHIIIANLYLLLPPTLNPIVYGVKTKQIREGVIKLFARQKVV Olfr669 DNA Sequence No. 19 atgctgatttccaacaactcatatgaagccccgcagtctttcattcttaatggaattcctggtctcgaagcagtgcatatatggatctctcttccactctgtacaatgtacatcatctccctagtaggcaaccttggccttgtatatctcatttactatgaggaatccttacatcgcccaatgtatttctttctggccatgctttctctcatagacctgtttacttgcacaaccactgtccccaatgccctcttcattttctggttcaaactcaaggaaattaacttcactgcttgcctagttcagatgttctttgtgcacggattcacaggtgtggagtctggggtactcatgctcatggccttggaccgctatgtggccatttgctacccactacgctatgcaaccatacttaccaaccctgtcattgccaaagctgggcttgccaccttcttgagaggtgtgttactgatgattccttttccattcttggttaaacgtttgcccttctgccgaagcaatgtcatctcccatacatattgtgaccacatgtctgtggtaaagttatcctgtgccagcatcaaaatcaatgtcatctatggtctcatggttgcacttctgattggagtgtttgacatatgttgtatatctgtgtcctacactatgatcctccgggcagtggtcagcctgtcctctgcagatgctcggcagaaggccttcagcacctgcacagcccacatatctgccatcatcattacttatgttccagccttcttcaccttctttactcatcgttttggaggtcacaccatccctccttctcttcatatcattgtggctaatctttatcttcttctccctccaactctaaatcccattgtttatgggatgaagaccaaacagatcagagatagtatcattaaattctttcacggtgaaaaaggttcaaggtga Protein - SEQ ID NO: 20 MLISNNSYEAPQSFILNGIPGLEAVHIWISLPLCTMYIISLVGNLGLVYLIYYEESLHRPMYFFLAMLSLIDLFTCTTTVPNALFIFWFKLKEINFTACLVQMFFVHGFTGVESGVLMLMALDRYVAICYPLRYATILTNPVIAKAGLATFLRGVLLMIPFPFLVKRLPFCRSNVISHTYCDHMSVVKLSCASIKINVIYGLMVALLIGVFDICCISVSYTMILRAVVSLSSADARQKAFSTCTAHISAIIITYVPAFFTFFTHRFGGHTIPPSLHIIVANLYLLLPPTLNPIVYGMKTKQIRDSIIKFFHGEKGSR Olfr1211 DNA sequence number 21 atgcaaaaccagagttttgtaacagaattcatattccttggactttcacagaaccctaaagtccagaaaatagtttttattgtatttttatttgtctacattgcaactgttgggggcaacatgataattgtggtgaccattgtctgtagcccagcattgatagactgccccatgtacttctttttggcattcttgtccctattggatgcatgcttctcttctgtcatcacaccaaagatggttgtggactccctgtatgagaagaaaactatctcctttgaaggatgtatgatgcagttatttgctgagcacttccttgcagcagtagaagtgattgtcttgacagccatggcctatgaccgctatgtagcaatttgcaagcccttgcactactcttccatcatgaactggaggctctgtggcacacttatggggatagcatggacagggggcttcttgcattctatcatacaaattatcttcacgttgcaattgcccttctgtggaccaaatgtcatcgatcatttcatgtgtgacttgttcccattactggaacttgcctgcactgatactcatatctttggccttttagtggttgccaacagtgggtctatctgcatcataatcttctctattttgctggtctcctatggtgtcatcctgttctctctgaaagctcacagttctgaagggcgatggaaagctctctccacatgtggatcccacattgcagttgtggttttgttctttgtcccgtgtatatttatttatgcacgtcctccatctgctttctcctttgataaaatggtggcgatattttatactatcctaactcccttgctcaatcctgtgatttatacttttcggaataaggacatgaaaaatgctatgaagaaagtgtggaagaggttggcagtggtttctgatggaaagtga Protein - SEQ ID NO: 22 MQNQSFVTEFIFLGLSQNPKVQKIVFIVFLFVYIATVGGNMIIVVTIVCSPALIDCPMYFFLAFLSLLDACFSSVITPKMVVDSLYEKKTISFEGCMMQLFAEHFLAAVEVIVLTAMAYDRYVAICKPLHYSSIMNWRLCGTLMGIAWTGGFLHSIIQIIFTLQLPFCGPNVIDHFMCDLFPLLELACTDTHIFGLLVVANSGSICIIIFSILLVSYGVILFSLKAHSSEGRWKALSTCGSHIAVVVLFFVPCIFIYARPPSAFSFDKMVAIFYTILTPLLNPVIYTFRNKDMKNAMKKVWKRLAVVSDGK OR52N5 DNA sequence number 23 atgcctctatttaattcattatgctggtttccaacaattcatgtgactcctccatcttttattcttaatggaatacctggtctggaaagagtacatgtatggatctccctcccactctgcacaatgtacatcatcttccttgtggggaatcttggtcttgtgtacctcatttattatgaggagtccttacatcatccgatgtattttttttttggccatgctctctccctcattgacctccttacctgcaccaccactctacccaatgcactctgcatcttctggttcagtctcaaagaaattaacttcaatgcttgcttggcccagatgttctttgttcatgggttcacaggtgtggagtctggggtgctcatgctcatggctctagaccgctatgtagccatttgctaccctttgcgttatgctaccacactcaccaaccctatcattgccaaggctgagcttgccaccttcctgaggggtgtattgctgatgattcctttcccattcttggttaagcgtttgcctttctgccaaagcaatattatctcccatacgtactgcgaccacatgtctgtagtaaagctatcttgtgccagcatcaaggtcaatgtaatctatggtctaatggttgctctcctgattggagtgtttgacatttgttgtatatctttgtcttacactttgatcctcaaggcagcgatcagcctctcttcatcagatgctcggcagaaggctttcagcacctgcactgcccatatatctgccatcatcatcacctatgttccagcattcttcactttctttgcccaccgttttgggggacacacaattcccccttctcttcacatcattgtggctaatctttatcttcttcttcccccaactctaaaccctattgtttatggagtaaagacaaaacagatacgcaagagtgtcataaagttcttccagggtgataagggtgcaggttga Protein - SEQ ID NO: 24 MPLFNSLCWFPTIHVTPPSFILNGIPGLERVHVWISLPLCTMYIIFLVGNLGLVYLIYYEESLHHPMYFFFGHALSLIDLLTCTTTLPNALCIFWFSLKEINFNACLAQMFFVHGFTGVESGVLMLMALDRYVAICYPLRYATTLTNPIIAKAELATFLRGVLLMIPFPFLVKRLPFCQSNIISHTYCDHMSVVKLSCASIKVNVIYGLMVALLIGVFDICCISLSYTLILKAAISLSSSDARQKAFSTCTAHISAIIITYVPAFFTFFAHRFGGHTIPPSLHIIVANLYLLLPPTLNPIVYGVKTKQIRKSVIKFFQGDKGAG OR2L13 DNA sequence number 25 atggagaaatggaatcacacttcaaatgatttcattttgttgggtctgcttcccccaaatcaaactggaatatttctcttgtgccttatcatcctcatattctttctggcctcggtgggtaactcggccatgattcacctcatccacgtggatcctcgtctccacacaccgatgtactttcttctcagccagctctcccttatggacctgatgtacatctccaccaccgtccccaagatggcgtacaacttcctgtccggccagaaaggcatctccttcctgggatgtggtgtgcaaagcttcttcttcctgaccatggcgtgttctgaaggcttactcctgacctccatggcctacgaccgttatttggccatctgccactctctctattatcctatccgcatgagtaaaatgatgtgtgtgaagatgattggaggctcttggacactggggtccatcaactccttggcacacacagtctttgcccttcatattccctactgcaggtctagggctattgaccatttcttctgcgatgtcccagccatgttgcttcttgcctgtacagatacttgggtctatgaatatatggtttttgtaagtacaagcctctttctccttttccctttcattggcatcacttcttcctgtggccgagtcctatttgctgtctatcatatgcactcaaaggaggggagaaaaaaggccttcaccaccatttcaacacatttaactgtagtgatcttttactatgcaccttttgtctacacctatcttcggcccaggaatctccgctcaccagctgaagacaagatcctggcagtcttctacaccatccttacccccatgctcaatcccattatctacagcctgaggaataaggaagtcctgggggctatgaggagagtgtttgggatattctctttcctgaaagaataa Protein - SEQ ID NO: 26 MEKWNHTSNDFILLGLLPPNQTGIFLLCLIILIFFLASVGNSAMIHLIHVDPRLHTPMYFLLSQLSLMDLMYISTTVPKMAYNFLSGQKGISFLGCGVQSFFFLTMACSEGLLLTSMAYDRYLAICHSLYYPIRMSKMMCVKMIGGSWTLGSINSLAHTVFALHIPYCRSRAIDHFFCDVPAMLLLACTDTWVYEYMVFVSTSLFLLFPFIGITSSCGRVLFAVYHMHSKEGRKKAFTTISTHLTVVIFYYAPFVYTYLRPRNLRSPAEDKILAVFYTILTPMLNPIIYSLRNKEVLGAMRRVFGIFSFLKE OR2AJ1 DNA Sequence No. 27 atgagtgtaacagaaaatacgctcatgatcctcctcattcgcagtgactcccgactccacactccaatgtattttctgctcagccatctctccttaatggatatcttgcatgtttccaacatcgttcccaaaatggtcactaactttctgtcaggcagcagaactatttcatttgcaggttgtgggttccaggtatttctgtccctcaccctcctgggtggtgagtgccttctcctggctgcaatgtcctgtgatcgctatgtggctatctgtcacccgctgcgctatccgattcttatgaaggagtatgccagcgctctcatggctggaggctcctggctcattggggttttcaactccacagtccacacagcttatgcactgcagtttcccttctgtggctctagggcaattgatcacttcttctgtgaagtccctgccatgttgaagttgtcctgtgcagacacaacacgctatgaacgaggggtttgtgtaagtgctgtgatcttcctgctgatccctttctccttgatctctgcttcttatggccaaattattcttactgtcctccagatgaaatcatcagaggcaaggaaaaagtcattttccacttgttccttccacatgattgtggtcacgatgtactatgggccatttatttttacatatatgagacctaaatcataccacactccagggcaggataagttcctggcaatattctatacgatcctcacacccacactcaaccctttcatctacagctttaggaataaagatgttctggcggtgatgaaaaatatgctcaaaagtaactttctgcacaaaaaaatgaataggaaaattcctgaatgtgtgttctgtctatttctatgttaa Protein - SEQ ID NO: 28 MSVTENTLMILLIRSDSRLHTPMYFLLSHLSLMDILHVSNIVPKMVTNFLSGSRTISFAGCGFQVFLSLTLLGGECLLLAAMSCDRYVAICHPLRYPILMKEYASALMAGGSWLIGVFNSTVHTAYALQFPFCGSRAIDHFFCEVPAMLKLSCADTTRYERGVCVSAVIFLLIPFSLISASYGQIILTVLQMKSSEARKKSFSTCSFHMIVVTMYYGPFIFTYMRPKSYHTPGQDKFLAIFYTILTPTLNPFIYSFRNKDVLAVMKNMLKSNFLHKKMNRKIPECVFCLFLC OR4C15 DNA sequence number 29 Protein - SEQ ID NO: 30 MFSMTTEALNNFALGCTNLLMTMIPQIDLKQIFLCPNCRLYMIPVGAFIFSLGNMQNQSFVTEFVLLGLSQNPNVQEIVFVVFLFVYIATVGGNMLIVVTILSSPALLVSPMYFFLGFLSFLDACFSSVITPKMIVDSLYVTKTISFEGCMMQLFAEHFFAGVEVIVLTAMAYDRYVAICKPLHYSSIMNRRLCGILMGVAWTGGLLHSMIQILFTFQLPFCGPNVINHFMCDLYPLLELACTDTHIFGLMVVINSGFICIINFSLLLVSYAVILLSLRTHSSEGRWKALSTCGSHIAVVILFFVPCIFVYTRPPSAFSLDKMAAIFYIILNPLLNPLIYTFRNKEVKQAMRRIWNRLMVVSDEKENIKL OR5AC2 DNA - SEQ ID NO: 31 atggatatatcagagggaaataagactcttgtgacagagtttgttctcacaggacttacagatcgaccatggctgcacgtcctcttctttgttgtgtttttggtggtctatctcatcaccatggtgggcaaccttggactgatagttctaatttggaacgacccccatcttcatatgcccatgtacttattccttggtggtttagccttttcagatgcttgtacttcaacctctataacccctaggatgctggtcaatttcttagacaagactgcaatgatatccctagctgagtgcatcacccagttttacttttttgcttccagtgcaactacagaatgcttcctcctggtgatgatggcctatgaccgctatgtagccatatgtaatcccttgctttatccagtgatgatgtccaacaaactcagcgctcagttgctaagtatttcatatgtaattggtttcctgcatcctctggttcatgtgagtttactattgcgactaactttctgcaggtttaacataatacattatttctactgtgaaattttacaactgttcaaaatttcatgcaatggtccatctattaacgcactaatgatatttatttttggtgcttttatacaaatacccactttaatgactatcataatctcttatactcgtgtgctctttgatattctgaaaaaaaagtctgaaaagggcagaagcaaagccttctccacatgcggcgcccatctgctttctgtctcattgtactacggaactctgatcttcatgtatgtgcgtcctgcatctggcttagctgaagaccaagacaaagtgtattctctgttttacacgattataattcccctgctaaacccatttatttacagcttgagaaataaaaaagtcatgcatgcattgagaagagttataaggaagtaa Protein - SEQ ID NO: 32 MDISEGNKTLVTEFVLTGLTDRPWLHVLFFVVFLVVYLITMVGNLGLIVLIWNDPHLHMPMYLFLGGLAFSDACTSTSITPRMLVNFLDKTAMISLAECITQFYFFASSATTECFLLVMMAYDRYVAICNPLLYPVMMSNKLSAQLLSISYVIGFLHPLVHVSLLLRLTFCRFNIIHYFYCEILQLFKISCNGPSINALMIFIFGAFIQIPTLMTIIISYTRVLFDILKKKSEKGRSKAFSTCGAHLLSVSLYYGTLIFMYVRPASGLAEDQDKVYSLFYTIIIPLLNPFIYSLRNKKVMHALRRVIRK OR8H3 DNA Sequence No. 33 atgatgggtagaaggaatgacacaaatgtggctgacttcatccttacgggactgtcagactctgaagaggtccagatggctctgtttatgctatttctcctcatatacctaattactatgctggggaatgtggggatgctattgataatccgcctggacctccagcttcacactcccatgtattttttccttactcacctgtcatttattgacctcagttactcaactgtcgtcacacctaaaaccttagcgaacttactgacttccaactatatttccttcacgggctgctttgcccagatgttctgttttgtcttcttgggtactgctgaatgttatcttctctcctcaatggcctatgatcgctatgcagcgatctgcagtcctctacactacacagttattatgcccaaaaggctctgcctcgctctcatcactgggccttatgtgattggctttatggactcctttgtcaatgtggtttccatgagcagattgcatttctgtgactcaaacataattcatcactttttctgtgacacttccccaattttagctctgtcctgcactgacacagacaacactgaaatgctgatattcattatcgctggttccaccctgatggtgtcccttatcacaatatctgcatcctatgtgtccattctctctaccatcctgaaaattaattccacttcaggaaagcagaaagctttctctacttgcgtctctcatctcttgggagtcaccatcttctatggaactatgatttttacttacttaaagccaagaaagtcttattccttgggaagagatcaagtggctcctgtgttttatactattgtgattcccatgctgaatccactcatttatagtcttagaaacagagaagtgaaaaatgctctcattagagtcatgcagagaagacaggactccaggtag Protein - SEQ ID NO: 34 MMGRRNDTNVADFILTGLSDSEEVQMALFMLFLLIYLITMLGNVGMLLIIRLDLQLHTPMYFFLTHLSFIDLSYSTVVTPKTLANLLTSNYISFTGCFAQMFCFVFLGTAECYLLSSMAYDRYAAICSPLHYTVIMPKRLCLALITGPYVIGFMDSFVNVVSMSRLHFCDSNIIHHFFCDTSPILALSCTDTDNTEMLIFIIAGSTLMVSLITISASYVSILSTILKINSTSGKQKAFSTCVSHLLGVTIFYGTMIFTYLKPRKSYSLGRDQVAPVFYTIVIPMLNPLIYSLRNREVKNALIRVMQRRQDSR OR11G2 DNA sequence number 35 atgaaaatcttcaacagccccagcaactccagcaccttcactggcttcatcctcctgggcttcccttgccccagggaggggcagatcctcctctttgtgctcttcactgttgtttacctcctgaccctcatgggcaatggttccatcatctgtgctgtgcactgggatcagagactccacgcccccatgtacatcctgctcgccaacttctccttcttggagatatgttatgtcacctccacagtccccagcatgctggccaacttcctctctgacaccaagatcatctcgttctctggctgcttcctccagttctactttttcttctccttgggctctacagaatgctttttcctggcagttatggcatttgatcgataccttgccatctgtcggcctctacgctatccaaccattatgaccagacgtctctgtaccaatcttgtggtcaattgctgggtacttggtttcatctggttcttgattcctatcgtcaacatctcccaaatgtccttctgtggatctaggattattgaccacttcctatgtgacccagctcctcttctaactctcacttgcaaaaaaggccctgtgatagagcttgtcttttctgtcttaagtcctctgcctgtctttatgctctttctcttcattgtggggtcctatgctctggtcgtgagagctgtgttgagggtcccttcagcagctgggagaagaaaggctttctccacctgtgggtctcacctggctgtggtttcactgttctacggctcagtactggtcatgtatgggagcccaccatctaagaatgaagctggaaagcagaagactgtgactctgttttattctgttgttaccccactgcttaaccctgtgatatatagtcttaggaacaaagatatgagaaaagctctgaagaaattttggggaacataa Protein - SEQ ID NO: 36 MKIFNSPSNSSTFTGFILLGFPCPREGQILLFVLFTVVYLLTLMGNGSIICAVHWDQRLHAPMYILLANFSFLEICYVTSTVPSMLANFLSDTKIISFSGCFLQFYFFFSLGSTECFFLAVMAFDRYLAICRPLRYPTIMTRRLCTNLVVNCWVLGFIWFLIPIVNISQMSFCGSRIIDHFLCDPAPLLTLTCKKGPVIELVFSVLSPLPVFMLFLFIVGSYALVVRAVLRVPSAAGRRKAFSTCGSHLAVVSLFYGSVLVMYGSPPSKNEAGKQKTVTLFYSVVTPLLNPVIYSLRNKDMRKALKKFWGT OR52N2 DNA Sequence No. 37 atgtctggggacaacagctccagcctgaccccaggattctttatcttgaatggcgttcctgggctggaagccacacacatctggatctccctgccattctgctttatgtacatcattgctgtcgtggggaactgtgggctcatctgcctcatcagccatgaggaggccctgcaccggcccatgtactacttcctggccctgctctccttcactgatgtcaccttgtgcaccaccatggtacctaatatgctgtgcatattctggttcaacctcaaggagattgactttaacgcctgcctggcccagatgttttttgtccatatgctgacagggatggagtctggggtgctcatgctcatggccctggaccgctatgtggccatctgctaccccttacgctatgccaccatccttaccaaccctgtcatcgccaaggctggtcttgccaccttcttgaggaatgtgatgctcatcatcccattcactctcctcaccaagcgcctgccctattgccgggggaacttcatcccccacacctactgtgaccatatgtctgtggccaaggtatcctgtggcaatttcaaggtcaatgctatttatggtctgatggttgctctcctgattggtgtgtttgatatctgctgtatctctgtatcttacactatgattttgcaggctgttatgagcctgtcatcagcagatgctcgtcacaaagccttcagcacctgcacatctcacatgtgttccattgtgatcacctatgttgctgcttttttcacttttttcactcatcgttttgtaggacacaatatcccaaaccacatacacatcatcgtggccaacctttatctgctactgcctcctaccatgaacccaattgtttatggagtcaagaccaagcagattcaggaaggtgtaattaaatttttacttggagacaaggttagttttacctatgacaaatga Protein - SEQ ID NO: 38 MSGDNSSSLTPGFFILNGVPGLEATHIWISLPFCFMYIIAVVGNCGLICLISHEEALHRPMYYFLALLSFTDVTLCTTMVPNMLCIFWFNLKEIDFNACLAQMFFVHMLTGMESGVLMLMALDRYVAICYPLRYATILTNPVIAKAGLATFLRNVMLIIPFTLLTKRLPYCRGNFIPHTYCDHMSVAKVSCGNFKVNAIYGLMVALLIGVFDICCISVSYTMILQAVMSLSSADARHKAFSTCTSHMCSIVITYVAAFFTFFTHRFVGHNIPNHIHIIVANLYLLLPPTMNPIVYGVKTKQIQEGVIKFLLGDKVSFTYDK OR5T1 DNA Sequence No. 39 atgtttatattaataagcttcacagaagaatttgatgtgcaagtcttcctatttttattatttttagcaatctatctattcactctaataggcaatttagggctggttgtaccgatcattggggatttctggcttcacagcccaatgtactattttcttggtgttttatcattcttggatgtctgctattctacagttgtcactccaaaaatgttggtcaatttcctggcaaaaaataaatctatttcatttcttggatgtgcaacacagatgtttcttgcttgtacttttggaaccacagaatgctttctcttggctgcaatggcttatgatcgctatgtagccatctacaaccctctcctgtattcagtgagcatgtcacccagagtctatgtgccactcatcactgcttcctatgttgctagcattttacatgctactatacatacagtggctacatttagcctgtccttctgtggatccaatgaaattaggcatgtcttttgtaatatgcctcctctccttgctatttcttgttctgacactcacgtaatccagcttctattcttctactttgtgggctctattgagatagtcactatcctgattgtcctgatctcctatggttttattctgttggccattctgaagatgcagtctgctgaagggaggagaaaagtcttctctacatgtggagctcacctaactggagtgacaatttatcatgggacaatcctcttcatgtatgtgagaccaagttccagctacacttcggacaatgacatgatagtgtcaatattttataccattgtgattcccatgctgaatcccatcatctacagtttgcggaacaaagatgtaaaggaggcaatcaaaagattgcttgtgagaaattggttcataaataagttatag Protein - SEQ ID NO: 40 MFILISFTEEFDVQVFLFLLFLAIYLFTLIGNLGLVVPIIGDFWLHSPMYYFLGVLSFLDVCYSTVVTPKMLVNFLAKNKSISFLGCATQMFLACTFGTTECFLLAAMAYDRYVAIYNPLLYSVSMSPRVYVPLITASYVASILHATIHTVATFSLSFCGSNEIRHVFCNMPPLLAISCSDTHVIQLLFFYFVGSIEIVTILIVLISYGFILLAILKMQSAEGRRKVFSTCGAHLTGVTIYHGTILFMYVRPSSSYTSDNDMIVSIFYTIVIPMLNPIIYSLRNKDVKEAIKRLLVRNWFINKL OR4S2 DNA sequence number 41 atggaaaaaataaacaacgtaactgaattcattttctggggtctttctcagagcccagagattgagaaagtttgttttgtggtgttttctttcttctacataatcattcttctgggaaatctcctcatcatgctgacagtttgcctgagcaacctgtttaagtcacccatgtatttctttctcagcttcttgtcttttgtggacatttgttactcttcagtcacagctcccaagatgattgttgacctgttagcaaaggacaaaaccatctcctatgtggggtgcatgttgcaactgtttggagtacatttctttggttgcactgagatcttcatccttactgtaatggcctatgatcgttatgtggctatctgtaaacccctacattatatgaccatcatgaaccgggagacatgcaataaaatgttattagggacgtgggtaggtgggttcttacactccattatccaagtggctctggtagtccaactacccttttgtggacccaatgagatagatcactacttttgtgatgttcaccctgtgttgaaacttgcctgcacagaaacatacattgttggtgttgttgtgacagccaacagtggtaccattgctctggggagttttgttatcttgctaatctcctacagcatcatcctagtttccctgagaaagcagtcagcagaaggcaggcgcaaagccctctccacctgtggctcccacattgccatggtcgttatctttttcggcccctgtacttttatgtacatgcgccctgatacgaccttttcagaggataagatggtggctgtattttacaccattatcactcccatgttaaatcctctgatttatacactgagaaatgcagaagtaaagaatgcaatgaagaaactgtggggcagaaatgttttcttggaggctaaagggaaatag Protein - SEQ ID NO: 42 MEKINNVTEFIFWGLSQSPEIEKVCFVVFSFFYIIILLGNLLIMLTVCLSNLFKSPMYFFLSFLSFVDICYSSVTAPKMIVDLLAKDKTISYVGCMLQLFGVHFFGCTEIFILTVMAYDRYVAICKPLHYMTIMNRETCNKMLLGTWVGGFLHSIIQVALVVQLPFCGPNEIDHYFCDVHPVLKLACTETYIVGVVVTANSGTIALGSFVILLISYSIILVSLRKQSAEGRRKALSTCGSHIAMVVIFFGPCTFMYMRPDTTFSEDKMVAVFYTIITPMLNPLIYTLRNAEVKNAMKKLWGRNVFLEAKGK FLAG (registered trademark) tag DNA - Sequence number 43 gattacaaggacgacgacgataag Protein - Sequence number 44 DYKDDDDK Rho tag DNA - Sequence number 45 atgaacgggaccgagggcccaaacttctacgtgcctttctccaacaagacgggcgtggtg Protein - Sequence number 46 MNGTEGPNFYVPFSNKTGVV Lucy tag DNA - Sequence number 47 atgagaccccagatcctgctgctcctggccctgctgaccctaggcctggct Protein - Sequence number 48 MRPQILLLLALLTLGLA Human G protein alpha subunit G olf DNA - Sequence number 49 Protein - SEQ ID NO: 50 MGLCYSLRPLLFGGPGDDPCAASEPPVEDAQPAPAPALAPVRAAARDTARTLLPRGGEGSPACARPKADKPKEKRQRTEQLSAEEREAAKEREAVKEARKVSRGIDRMLRDQKRDLQQTHRLLLLGAGESGKSTIVKQMRILHVNGFNPEEKKQKILDIRKNVKDAIVTIVSAMSTIIPPVPLANPENQFRSDYIKSIAPITDFEYSQEFFDHVKKLWDDEGVKACFERSNEYQLIDCAQYFLERIDSVSLVDYTPTDQDLLRCRVLTSGIFETRFQVDKVNFHMFDVGGQRDERRKWIQCFNDVTAIIYVAACSSYNMVIREDNNTNRLRESLDLFESIWNNRWLRTISIILFLNKQDMLAEKVLAGKSKIEDYFPEYANYTVPEDATPDAGEDPKVTRAKFFIRDLFLRISTATGDGKHYCYPHFTCAVDTENIRRVFNDCRDIIQRMHLKQYELL Human G - protein alpha subunit Gα15 DNA - SEQ ID NO: 51 Protein - SEQ ID NO: 52 MARSLTWRCCPWCLTEDEKAAARVDQEINRILLEQKKQDRGELKLLLLGPGESGKSTFIKQMRIIHGAGYSEEERKGFRPLVYQNIFVSMRAMIEAMERLQIPFSRPESKHHASLVMSQDPYKVTTFEKRYAAAMQWLWRDAGIRACYERRREFHLLDSAVYYLSHLERITEEGYVPTAQDVLRSRMPTTGINEYCFSVQKTNLRIVDVGGQKSERKKWIHCFENVIALIYLASLSEYDQCLEENNQENRMKESLALFGTILELPWFKSTSVILFLNKTDILEEKIPTSHLATYFPSFQGPKQDAEAAKRFILDMYTRMYTGCVDGPEGSKKGARSRRLFSHYTCATDTQNIRKVFKDVRDSVLARYLDEINLL SEQ ID NO: 53 Motif MAYDRYVAIC SEQ ID NO: 54 Motif FSTCSSH SEQ ID NO: 55 Motif PMLNPFIY

[0088] The following examples are for illustrative purposes only and do not limit the scope of the invention described in the summary of the invention, detailed description, or claims.

Brief Description of the Drawings

[0089]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0090] Example Example 1 Identification of Novel Mouse and Human DMTS-Activated Odorant Receptors The identification of new odorant receptors was carried out according to the method disclosed in International Publication No. WO 2014 / 210585. Briefly, mouse olfactory neurons were exposed to DMTS, Ca 2+Screening was performed using imaging technology. Neurons activated by DMTS were further isolated for complete transcriptome analysis to identify responsive odorant receptors. cDNA corresponding to the isolated cellular mRNA was generated and amplified by a PCR-based method (Clontech / Takara, SMARTer® Ultra® Low Input RNA Kit for Sequencing-v3, cat.634848). Next, the amplified cDNA was used to generate an Illumina cDNA library for next-generation sequencing and the generation of 100-base pair single-read sequences. The sequences were aligned to the mouse reference genome (e.g., UCSC version mm10) to generate a complete transcriptome. Since only one olfactory receptor (OR) is strongly transcribed per olfactory neuron, subsequent identification of DMTS-responsive ORs can be achieved. Next, corresponding human ORs were identified using phylogenetic relationship assessment with sequence similarity searches. Similar functional response profiles are often observed between orthologous OR pairs [e.g., Adipietro, K. A et al., PLoS Genet. 8, e1002821–e1002821 (2012), Sato-Akuhara, N. et al., J. Neurosci. 36, 4482–4491 (2016) and International Publication No. 2016 / 201152], and can be used for human OR identification [e.g., Armelin-Correa L. M. and Malnic B. J Agric Food Chem. doi: 10. 1021 / acs. jafc. 6b04998 (2017)]. Figure 1 shows the identity levels for pairs of receptors mentioned herein. ORs that do not share orthologous and / or paralogous relationships share only 39% ± 13.1% on average. All orthologous and paralogous relationships share 77% ± 7.5% on average. All paralogous and orthologous pairs are shown in gray-shaded cells. They represent the highest identity levels supported by the indicated amino acid identity levels.

[0091] Example 2 Functional Characterization of Mouse and Human DMTS Receptors To evaluate the level of DMTS activity of modified cell lines expressing individual putative DMTS receptors, functional dose-response experiments were performed. Using a cell-based assay, mouse receptors Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738 and Olfr742, as well as human receptors OR4S2 and OR52N5, were tested in the HEK293T cell line, activating the endogenous RTP1 gene and expressing a chaperone protein for odorant receptors (described in International Publication No. 2016 / 201153). Mouse receptor genes were tagged with a combination of Lucy-FLAG (registered trademark)-Rho tags, and human receptors were tagged with a combination of Rho-FLAG (registered trademark) tags, resulting in tag::receptor fusion proteins in the cell-based assay. The receptor genes were co-transfected with the standard olfactory human G protein alpha subunit G olf gene and exposed to increasing concentrations of malodorous odorants. Co-expression of human G olf activates the Gs signaling pathway that leads to an increase in intracellular cAMP upon binding to the appropriate ligand. Odorant-induced activity was detected by measuring the increase in cytosolic cAMP using a homogeneous time-resolved fluorescence (HTRF)-based kit (CisBio, cAMP dynamic 2 kit, cat.62AM4PEJ). A dose-dependent increase in receptor activity was specifically seen for DMTS but not for butyric acid, another known malodor used as a negative control (Figure 1). Activity levels are reported by the potency of the DMTS-induced response of each receptor as a measure of the EC50 value (the effective concentration at which the receptor responds at half the level of the maximum activation effect of a given compound).

[0092] Example 3 Functional Characterization of Additional Human DMTS Receptors To evaluate the level of DMTS activity of modified cell lines expressing individual putative DMTS receptors, functional dose-response experiments were performed. Cell lines stably expressing human receptors were generated in the HEK293T cell line for each of the following human receptors: OR2L13, OR4C15, OR5AC2, OR8H3, OR11G2 or OR52N2. The receptors were tagged with a combination of FLAG®-Rho tags and stably co-expressed with the human G protein alpha subunit Gα 15 The co-expression of human Gα 15 activates the Gq signaling pathway leading to an increase in intracellular Ca 2+ upon binding to the appropriate ligand. The receptors were exposed to increasing concentrations of DMTS. The increase in cytosolic Ca 2+ was measured using a calcium-sensitive fluorescent dye (Molecular Devices, Calcium 5 dye, cat. R8186), and odorant-induced activity was detected by measuring the change in relative fluorescence ratio (RFU) using a fluorescence imaging plate reader (Molecular Devices, FLIPR) after exposure to the odorant. The dose-dependent increase in receptor activity was recorded and the corresponding dose-response curves are shown for DMTS (Figure 2). A cell line lacking the receptor was used as a control for non-specific activity at high compound concentrations ("receptorless"). The activity levels are reported by the potency of the DMTS-induced response of each receptor as a measure of the EC50 value.

[0093] Example 4 Identification of DMTS Receptor Inhibitors The stable cell lines described in Example 3 were used as an antagonist screening platform to identify compounds with properties that reduce DMTS-induced receptor activity. Each stable cell line expressing a human odorant receptor was screened with a volatile compound library for its inhibitory properties and potential suppression of DMTS odor. First, individual binary mixtures of DMTS and each test compound were presented to the cells. Single-point monitoring of DMTS-induced cell activity in the presence or absence of the test compound enabled the identification of compounds with putative inhibitory or suppressive effects. These hits were further confirmed in an inhibitory dose-response curve assay that evaluates the potency of activity inhibition using the IC50 (inhibitor concentration at which receptor activity is inhibited at half the level of the maximum inhibitory effect of a given test compound) as a measure. The dose-dependent decrease in receptor activity was recorded by increasing the concentration of the test compound in the presence of a single activating concentration of DMTS (EC80) to obtain a corresponding dose-response inhibition curve. The compounds in the table below are examples of compounds that decreased the DMTS-induced activity of at least one receptor, as shown in Figure 4.

Table 2

[0094] In Figure 4, the bar length indicates the potency of activity inhibition (expressed as IC50, negative logarithm of molar concentration) of the selected compounds for each receptor. The absence of a bar indicates no inhibition for the corresponding receptor-compound pair. These compounds can specifically bind to and block the activity of the DMTS or DMDS receptor and thus can suppress or inhibit the odor of DMTS or DMDS. Therefore, such compounds can be used as malodor neutralizers for toilet and bad breath control applications.

[0095] Example 5 Response profiles of mouse and human DMTS receptors to DMDS Functional dose-response experiments were performed to confirm the DMTS activity against the DMTS receptors identified in Example 2 and further characterize their responses to DMDS. Using the same cell-based assay described in Example 2, mouse receptors Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, and the human odorant receptor OR4S2 were tested with increasing concentrations of DMTS, DMDS, or butyric acid (control). A strong dose-dependent increase in receptor activity was observed for DMTS and DMDS relative to the control compound butyric acid. Weak responses of OR4S2 and Olfr169 to butyric acid were seen, but butyric acid was too weak to be considered a representative ligand for these receptors. The activity levels were reported as a measure of EC50 value, EC50 DMTS and EC50 DMDS for the potency of the DMTS or DMDS-induced response of each receptor, respectively. In mock transfection control experiments where cells did not express odorant receptors, no activity was shown upon exposure to DMTS or DMDS. Competitive antagonists at the malodor binding sites of these receptors may reduce the unpleasant perception of both DMTS and DMDS.

Claims

1. A method for identifying a compound that binds to, inhibits, blocks, inhibits, and / or regulates the activity of an olfactory receptor activated by a malodor-causing substance, comprising: a. contacting a test substance and a malodor-causing substance with at least one olfactory receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1; b. measuring the response of the olfactory receptor in the presence and absence of the test substance to measure the response of the olfactory receptor to the malodor-causing substance; c. identifying a test substance that modulates the response of the olfactory receptor based on the responses measured in the presence and absence of the test substance; and d. selecting the identified test substance as a compound that modulates the response of the olfactory receptor to the malodor-causing substance wherein, the malodor-causing substance is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

2. A method for identifying a malodor inhibitor, comprising: a. contacting a test substance and a malodor-causing substance with at least one olfactory receptor, wherein the receptor comprises a polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40; b. measuring the response of the olfactory receptor polypeptide to the malodor-causing substance; c. identifying a test substance that can inhibit the response of the olfactory receptor based on the measured response; and d. selecting the test substance that inhibits the response of the olfactory receptor as a malodor inhibitor wherein, A method wherein the substance causing the bad odor is DMTS or DMDS.

3. A method for identifying a compound that binds, inhibits, blocks, inhibits, and / or regulates the activity of at least one olfactory receptor activated by a substance causing a bad odor, comprising: a. contacting the receptor, or a chimera or fragment thereof, with a compound; and b. determining whether the compound affects the activity of the receptor wherein the receptor is i) a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40; or ii) a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39 or its reverse complement, wherein the substance causing the bad odor is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

4. A method for identifying a compound that presumptively regulates DMTS- or DMDS-related bad odor, comprising: (i) contacting a cell line expressing a DMTS or DMDS receptor polypeptide with at least one compound; (ii) screening for a compound that binds, inhibits, blocks, inhibits, and / or regulates the activity of the olfactory receptor polypeptide; and (iii) identifying a compound that presumptively regulates DMTS- or DMDS-related bad odor if the compound binds, inhibits, blocks, inhibits, and / or regulates the activity of the DMTS or DMDS receptor polypeptide, wherein the receptor polypeptide is a. comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40; or b. a method encoded by a nucleic acid molecule comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39 or its reverse complement. **Claim 5** An isolated polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO:

40. **Claim 6** An isolated nucleic acid molecule, a. a nucleic acid sequence encoding the polypeptide according to claim 5; or b. a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39 or its reverse complement An isolated nucleic acid molecule comprising. **Claim 7** A recombinant nucleic acid molecule, a. a nucleic acid comprising a combination of tags comprising at least one of a Lucy tag, a FLAG (registered trademark) tag, and / or a Rho tag; and b. A nucleic acid encoding a receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR4S2, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1 or a complement thereof A recombinant nucleic acid molecule comprising the same.

8. The recombinant nucleic acid molecule according to claim 7, wherein the Lucy tag comprises SEQ ID NO: 47, the FLAG (registered trademark) tag comprises SEQ ID NO: 43, and the Rho tag comprises SEQ ID NO:

45.

9. An expression vector comprising the nucleic acid according to any one of claims 6 to 8.

10. A non-human host organism or host cell modified to express a receptor activated by DMTS or DMDS, wherein the receptor is a. Selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, Olfr207, Olfr665, Olfr669, Olfr1211, OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, and OR5T1, or b. The polypeptide according to claim 5 or a polypeptide encoded by the nucleic acid according to any one of claims 6 to 8 A non-human host organism or host cell comprising the same.

11. A non-human host organism or host cell, wherein a. The nucleic acid according to any one of claims 6 to 8; or b. The expression vector according to claim 9 A non-human host organism or host cell comprising the same.

12. The non-human host organism or host cell according to claim 10 or 11, wherein the cell is a eukaryotic cell.

13. The non-human host organism or host cell according to claim 10 or 11, wherein the cell is a prokaryotic cell.

14. The non-human host organism or host cell according to claim 10 or 11, wherein the non-human host organism or host cell is selected from the group consisting of HEK293, CHO, African clawed frog oocytes, COS, yeast, and cells derived from olfactory placodes.

15. Use of a polypeptide that can be activated by DMTS or DMDS, comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40, for identifying an odor control compound.

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