Methods for identifying malodor-modulating compounds

Abstract

Methods are provided for identifying compounds that bind to, inhibit, block, inhibit, and / or modulate the activity of olfactory receptors activated by substances that cause malodors. [Solution] Provided are methods for identifying compounds that bind to, suppress, block, inhibit, and / or modulate the activity of one or more olfactory receptors activated by dimethyl trisulfide (DMTS), a substance that causes malodor, comprising: a) contacting a receptor, or a chimera or fragment thereof, with a compound; and b) determining whether the compound affects the activity of the receptor. Further provided are expression vectors containing nucleic acids encoding the described polypeptides, as well as non-human organisms or host cells modified to express DMTS-activated receptors. Also provided are uses of the polypeptides for identifying malodor-modulating compounds.

Description

[Technical field]

[0001] TECHNICAL FIELD TO WHICH THEINVENTION BELONGS The present technical field relates to odorants and aroma receptors, and assays that can be used to identify inhibitors, modulators or neutralizers of odorants and / or aroma compounds, more specifically malodorous compounds such as dimethyl trisulfide (DMTS).

[0002] background Olfaction is one of the most complex and poorly understood of the human sensory systems. From olfactory receptor (OR) activation to perception, there are many steps that still require further investigation. If we can understand how the OR codes of individual odorants and mixtures are translated into perception, this knowledge can be exploited to bring great benefits in several fields. These fields include odor modifiers such as malodor neutralizers that block the perception of unpleasant odors, new flavor and fragrance ingredients that replace non-biodegradable or toxic compounds, and odor enhancers that limit the reliance on compounds that are difficult to source 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 about 1,200 distinct intact ORs; in contrast, humans have about 400. In both cases, the repertoire of ORs is activated by thousands of odorants worldwide, and this combinatorial complexity allows for the breadth of perceptible olfaction. However, as of 2014, odorants or ligands have only been identified for 82 mouse ORs (~8%) and 17 human ORs (~10%) using traditional deorphanization methods. Moreover, the physiological relevance of most ligands for the human ORs essentially identified in vitro has never been tested.

[0003] A method that allows rapid and reliable identification of a relatively small subset of ORs within the total repertoire of ORs present in an organism that are specifically activated or inhibited by one or more odorants is described in WO 2014 / 210585. However, when using this method, it is necessary to identify the odorant receptors, and more specifically, the malodor receptors that are activated by the substances responsible for a particular malodor.

[0004] Malodor-causing compounds such as dimethyl trisulfide (DMTS) and other closely related polysulfide compounds such as dimethyl disulfide (DMDS) can produce unpleasant odors, for example, resulting from lavatories and other "toilet" sources containing feces or from bad breath. Thus, malodor regulators or neutralizers that bind, suppress, block, inhibit, and / or modulate the activity of one or more olfactory receptors activated by specific malodor-causing substances such as DMTS or DMDS are desirable. Further desirable are assays to identify new compounds or compound mixtures that rely on such new or newly identified malodor receptors associated with specific malodor-causing substances and bind to these receptors.

[0005] Summary of the Invention Provided herein is a non-human host organism or host cell modified to express a DMTS-activated receptor, wherein the receptor 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] Further provided is a non-human host organism or host cell 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.

[0007] Further 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 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.

[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 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 a reverse complement thereof.

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

[0010] Further provided herein is a method for identifying a compound that binds to, inhibits, blocks, inhibits and / or modulates the activity of at least one olfactory receptor activated by a malodor-causing substance, such as dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS), wherein said 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 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, said method comprising: a) contacting the receptor or chimera or fragment thereof with a compound; b) To determine whether the compound affects the activity of the receptor. A method is provided, comprising:

[0011] Also provided is a method for identifying compounds that bind, inhibit, block, inhibit, and / or modulate the activity of at least one olfactory receptor activated by a substance causing a malodor, comprising the steps of: a. contacting a test substance and a substance causing a malodor 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 olfactory receptors to substances causing malodours by measuring the response of olfactory receptors in the presence and absence of a test substance; c. identifying a test substance that modulates the response of an olfactory receptor based on the response measured in the presence and absence of the test substance; and d. Selecting the identified test substances as compounds that modulate the response of olfactory receptors to substances that cause malodours. Including, There is also provided a method, wherein the substance causing the malodor is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

[0012] Further provided is a method for identifying a malodor control agent, comprising the steps of: a. contacting the test substance and the malodor-causing substance with at least one olfactory receptor, wherein the receptor comprises 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; b. measuring the response of olfactory receptor polypeptides to substances that cause malodours; c. identifying test substances capable of inhibiting the response of the olfactory receptor based on the measured response; and d. Selecting test substances that bind, inhibit, block, inhibit, and / or modulate the response of olfactory receptors as malodor modulating agents. Including, Here, a method is provided, wherein the substance causing the malodor is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

[0013] Further provided is a recombinant nucleic acid molecule comprising a nucleic acid comprising at least one of a Lucy tag, a FLAG® 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 their reverse complements.

[0014] Still further provided are non-human host organisms or host cells recombinantly engineered to express the above-described nucleic acids or polypeptides.

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

[0016] Further, where the description of various embodiments uses the term "comprising," it should be understood that those skilled in the art will understand that in some specific instances the embodiments can alternatively be described using the words "consisting essentially of" or "consisting of."

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

[0018] "OR" refers to one or more members of a family of G protein-coupled receptors (GPCRs) 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 can 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 expressed in olfactory cells, which are bound and / or activated by DMTS or DMDS in a binding or activity assay to identify ligands that bind and / or activate GPCRs. Such assays are described below. DMTS or DMDS receptors herein are meant to include fragments, mutants, and chimeric or recombinant nucleic acids or proteins that respond to or bind to DMTS or DMDS, including synthetic and naturally occurring ones.

[0020] "OR" polypeptides are considered as such because they belong to the seven-transmembrane domain G protein-coupled receptor superfamily, encoded by a single approximately 1 kb long exon, and exhibit characteristic olfactory receptor-specific amino acid motifs. The seven domains are termed "transmembrane" or "TM" domains, TM I through TM VII, and are connected by three "inner cell loop" or "IC" domains, IC I through IC III, and three "outer cell loop" or "EC" domains, EC I through EC III. The motifs and variants thereof are defined as, but are not limited to, the MAYDRYVAIC motif (SEQ ID NO: 53) that overlaps with TM III and IC II, the FSTCSSH motif (SEQ ID NO: 54) that overlaps with IC III and TM VI, the PMLNPFIY motif (SEQ ID NO: 55) in TM VII and the presence of three conserved C residues in EC II, and the highly conserved GN residue in TMI [Zhang, X. & Firestein, S. Nat. Neurosci. 5, 124-133 (2002); Malnic, B. et al. Proc. Natl. Acad. Sci. USA 101, 2584-2589 (2004)].

[0021] "OR" nucleic acids, for example, encode a family of GPCRs with seven transmembrane domains that have "G protein-coupled receptor activity," which bind to G proteins in response to extracellular stimuli and mediate the production of IP3, cAMP, cGMP, Ca2+, and / or ATP through the stimulation of enzymes such as phospholipase C and adenylate cyclase. 2+ It may promote the production of second messengers such as

[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 phylogenetically linked by a gene present in a common ancestor and refer to closely related homologous genes in other species.

[0023] The "N-terminal domain" region begins at the N-terminus and extends to the region proximal to the beginning of the first transmembrane region. The "transmembrane region" includes the seven "transmembrane domains", which refers to the domains of the OR polypeptide that are within the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. The seven transmembrane regions and 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 as described in 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 the known transmembrane domain sequences, as described in detail below. These transmembrane domains are useful for in vitro ligand binding assays.

[0024] The phrase "functional effect" in the context of assays 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, e.g., functional, physical and chemical effects, such as ligand binding, changes in ion flux, membrane potential, current flow, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, signaling receptor-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP, IP3, or intracellular Ca), in vitro, in vivo and ex vivo. 2+ ), as well as other physiological effects such as an increase or decrease in neurotransmitter or hormone release.

[0025] "Measuring functional effects" or "confirming activity" in the context of an assay refers to assaying a compound to increase or decrease indirect or direct parameters under the influence of an OR family member, such as functional, physical and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, such as 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 currents, radioisotope efflux, inducible markers, oocyte OR gene expression; tissue culture cell OR expression; transcriptional activation of OR genes or activity-inducible genes such as egr-1 or c-fos; ligand binding assays; changes in voltage, membrane potential and conductance; ion flux assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; release of neurotransmitters, and the like.

[0026] "Binders," "suppressors," "blockers," "inhibitors," "activators," and / or "modulators" of OR genes or proteins are used interchangeably to refer to binding, suppressing, blocking, inhibiting, activating, or modulating molecules, e.g., ligands, agonists, antagonists, enhancers, and their homologs and mimetics, identified using in vivo, in vitro, and ex vivo assays for olfactory transduction. Inhibitors are, for example, compounds that bind, partially or completely block stimulation, reduce, inhibit, prevent, delay activation, inactivate, desensitize, or downregulate olfactory transduction, e.g., antagonists. Activators are, for example, compounds that bind, stimulate, increase, promote activation, promote, enhance activation, sensitize, or upregulate olfactory transduction, e.g., agonists. Modulators include, for example, compounds that modify the interaction of receptors with extracellular proteins that bind activators or inhibitors (e.g., odorant-binding proteins, Ebnerin and other members of the hydrophobic carrier family, or members of the lipocalin family); G proteins; kinases (e.g., homologs of rhodopsin kinase and beta-adrenergic receptor kinase, which are involved in receptor deactivation and desensitization); and arrestins, which further inactivate and desensitize receptors. Modulators also include compounds that modify the affinity or transduction efficiency of ORs and modify the effect of activators on ORs. Modulators can include, for example, genetically modified versions of OR family members with altered activity, as well as natural and synthetic ligands, antagonists, agonists, small chemical molecules, and the like. Assays for such inhibitors and activators include, for example, expressing an OR family member in a cell or cell membrane, applying a putative modulator compound in the presence or absence of a flavor, fragrance or malodorous molecule, e.g., a substance that causes a malodor, such as DMTS, and measuring the functional effect on olfactory transduction, as described above.Samples or assays containing OR family members treated with potential activators, inhibitors, or modulators are compared to control samples that do not contain the inhibitors, activators, or modulators to examine the degree of modulation. Control samples (untreated with modulators) are assigned a relative OR activity value of 100%. Inhibition of an OR is achieved when the OR activity value relative to the control is approximately 80%, optionally 50% or 25-0%.

[0027] The terms "purified", "substantially purified" and "isolated" as used herein refer to a state in which the compound of the invention is free of other distinct compounds with which it is normally associated in its natural state, and a "purified", "substantially purified" and "isolated" subject comprises at least 0.5%, 1%, 5%, 10% or 20% by weight of a given sample, most preferably at least 50% or 75% by weight of the mass of the given sample. In one preferred embodiment, these terms refer to a compound of the invention comprising 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" and "isolated" and "(substantially) isolated" for a nucleic acid or protein also refer to a state of purification or concentration different from that which naturally occurs in a mammal, particularly a human body. Any degree of purification or concentration greater than that which naturally occurs in a mammal, particularly a human body, including (1) purification from other related structures or compounds, or (2) association with structures or compounds with which it is not normally associated in the mammal, particularly a human body, falls within the meaning of "isolated". The nucleic acids or proteins or classes of nucleic acids or proteins described herein may be isolated or otherwise associated with structures or compounds with which they are not normally associated in nature, according to a variety of 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 clear to those skilled in the art that variants also include DNA sequence polymorphisms that may exist in a given population, which may lead to changes in the amino acid sequence of the polypeptide disclosed herein.Such genetic polymorphisms may exist in different populations of cells or populations due to natural allelic variation.Allelic variants may also include functional equivalents.

[0032] Further embodiments also relate to molecules derived from such sequence polymorphisms from the specifically disclosed nucleic acids. These natural variations usually result in about 1-5% variation in the nucleotide sequence of the gene or amino acid sequence of the polypeptides disclosed herein. As noted 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 contains natural amino acids or polymers and 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, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., 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] A "promoter" is defined as a sequence of nucleic acid sequences that directs the transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as a TATA element in the case of a polymerase II type promoter. A promoter also optionally includes distal enhancer or repression elements, which may be several thousand 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 the functional association 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 or otherwise manipulated in vitro, a method of using a recombinant polynucleotide to produce a gene product in a cell or other biological system, or a polypeptide (a "recombinant protein") encoded by a recombinant polynucleotide. "Recombinant" also refers to the ligation of nucleic acids with various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for inducible or constitutive expression, e.g., expression of a fusion protein containing a nucleic acid sequence potentially amplified using a translocation domain and primers of the invention. "Recombinant" also refers to the modification of a cell resulting in stable or transient expression of an endogenous gene, such as a receptor gene referred to herein, obtained by genome editing techniques such as CRISPR / Cas9.

[0036] The term "expression vector" refers to a recombinant expression system for constitutively or inducibly expressing the nucleic acid sequence of the present invention in any cell, such as prokaryotic, yeast, fungal, plant, insect or mammalian cells, in vitro, ex vivo, or in vivo. The term includes linear or circular expression systems, including but not limited to viral vectors, bacteriophages and plasmids. A person skilled in the art can select an appropriate vector according to the expression system. The term includes expression systems that remain episomal or are integrated into the host cell genome. The expression system may or may not be self-replicating, i.e., capable of promoting transient expression in the cell. The term includes recombinant "expression cassettes" that may contain the minimum elements required for transcription of recombinant nucleic acid. The term also covers cassettes or vectors for expression of endogenous genes via genome editing methods, such as CRISPR / Cas9.

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

[0038] A "tag" or "tag combination" refers to a short polypeptide sequence that can be added to an odorant receptor protein. Typically, DNA encoding a "tag" or a "tag combination" is added to DNA encoding a receptor, ultimately resulting in a fusion protein in which the "tag" or "tag combination" is fused to the N-terminus or C-terminus of the receptor. Lucy, FLAG® and / or Rho tags can enhance receptor trafficking to the cell membrane, thus aiding 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, HJ Biol. Chem. 282, 15284-15293 (2007)].

[0039] In one embodiment herein, an isolated nucleic acid molecule is provided comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity 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 a reverse complement thereof.

[0040] In one embodiment of the present specification, there is provided an isolated nucleic acid sequence as described above that encodes 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.

[0041] In further embodiments herein there is provided 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 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 an amino acid sequence identical 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.

[0044] Further 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 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.

[0045] Also provided herein is 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 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.

[0046] Also provided herein is 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 a reverse complement thereof.

[0047] In one embodiment herein, there is provided a method for identifying a compound that binds to, inhibits, blocks, inhibits, and / or modulates the activity of an 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 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, the method comprising: a) contacting the receptor or chimera or fragment thereof with a compound; b) To determine whether the compound affects the activity of the receptor. A method is provided, comprising:

[0048] In one embodiment, a method for identifying compounds that bind, inhibit, block, inhibit, and / or modulate the activity of olfactory receptors activated by substances that cause malodor is provided, comprising: a. contacting the test substance and the 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 olfactory receptors to substances causing malodours by measuring the response of olfactory receptors in the presence and absence of a test substance; c. identifying a test substance that modulates the response of an olfactory receptor based on the response measured in the presence and absence of the test substance; and d. Selecting the identified test substances as compounds that modulate the response of olfactory receptors to substances that cause malodours. Including, Here, a method is provided, wherein the substance causing the malodor is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

[0049] In a further embodiment herein there is provided a method for identifying a malodor control agent comprising: a. contacting the test substance and the malodor-causing substance with at least one olfactory receptor comprising 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; b. measuring the response of olfactory receptor polypeptides to substances that cause malodours; c. identifying test substances capable of modulating or inhibiting the response of olfactory receptors based on the measured responses; and d. Selecting test substances as malodor modulating agents that bind, inhibit, block, inhibit, and / or modulate the response of olfactory receptors. Including, Here, a method is provided, wherein the substance causing the malodor is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

[0050] One embodiment is a method for identifying a malodor control agent, comprising: a. contacting the test substance and the malodor-causing substance with at least one olfactory receptor comprising 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; b. measuring the response of olfactory receptor polypeptides to substances that cause malodours; c. identifying test substances capable of inhibiting 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 an odor suppressant. Including, The method according to the present invention is characterized in that the substance causing the malodor is DMTS and the malodor is fecal malodor or oral malodor.

[0051] One embodiment is a method for identifying a compound that putatively modulates DMTS-related malodors, 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 compounds that bind, inhibit, block, inhibit, and / or modulate the activity of the olfactory receptor polypeptide; and (iii) identifying a compound that putatively modulates DMTS-related malodors if it binds, inhibits, block, inhibit, and / or modulates the activity of the DMTS receptor polypeptide.

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

[0053] moreover, a. a nucleic acid comprising a combination of tags including one or more of Lucy, FLAG®, and / or Rho tags; 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:

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

[0055] Further provided are any of a number of malodor modulating compounds that bind to, inhibit, block, inhibit, and / or modulate the activity of olfactory receptors that are activated by malodor-causing substances, such as dimethyl trisulfide (DMTS), and identified by the methods disclosed herein.

[0056] In one embodiment herein, there is provided a malodor modulating compound that binds to, inhibits, 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 or 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 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 malodor modulating compounds that bind, inhibit, block, inhibit, and / or modulate the activity of olfactory receptors.

[0058] Still further, there is provided a cell recombinantly engineered to express the above-described polypeptide.

[0059] In one embodiment herein, there is provided a non-human host organism or host cell that has been transformed or modified to express a DMTS-activated 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.

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

[0061] In further embodiments 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 cell provided herein is a prokaryotic cell. In another embodiment, the cell provided herein is a eukaryotic cell. In a particular embodiment, the cell provided herein is selected from the group consisting of yeast cells and plant cells. In a more particular embodiment, the cell provided herein is selected from the group consisting of HEK293, CHO, Xenopus oocyte, COS, yeast, bacteria, and cells derived from the olfactory placode.

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

[0064] In one aspect of the present invention, methods are provided for identifying mammalian odorant receptors for malodor-modulating compounds and the use of the receptors for screening, particularly for high-throughput screening (HTS) of malodor-modulating agents (e.g., that bind, inhibit, block, inhibit, and / or modulate the activity of the OR).

[0065] In particular, 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 herein as receptors for the malodor-causing substances DMTS, DMDS, or other polysulfide compounds, as shown in Table 1. [Table 1]

[0066] Without wishing to be bound by theory, mouse receptor Olfr738 is a paralog of receptor Olfr742, and human OR11G2 is an orthologue of these mouse receptors; and mouse receptor Olfr669 is a paralog of receptor Olfr665 and an orthologue of human receptor OR52N5; and mouse receptor Olfr665 is an orthologue of human receptor OR52N2. Some receptors were previously identified as indole and / or skatole-responsive odorant receptors in WO 2014 / 210585. However, these receptors have not previously been associated with the substances DMTS or DMDS or other related polysulfide compounds that cause malodor. Indole and skatole are not chemically related to polysulfide compounds, so chemical structure-based approaches such as structure-activity relationship (SAR) to identify additional ligands would not define DMTS as a potential agonist for these receptors.

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

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

[0069] In a further embodiment, molecular 3D receptor modeling of olfactory receptors is used to evaluate in silico binding capabilities 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 glass microelectrodes attached to a micromanipulator or a FACS machine. Mouse olfactory neurons are isolated using Ca ion channels similar to the procedure previously described. 2+ The cells are screened by imaging [Malnic, B. et al., Cell 96, 713-723 (1999); Araneda, RC et al., J. Physiol. 555, 743-756 (2004); and WO 2014 / 210585, the entirety of which is incorporated herein by reference]. In particular, a motorized movable microscope stage is used to increase the number of cells that can be screened to at least 1,500 per experiment. Since mice have about 1,200 different olfactory receptors and each olfactory neuron expresses only one of the 1,200 olfactory receptor genes, this screening capacity will essentially cover the entire odorant receptor repertoire of mice. 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 specific 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] For calcium imaging of olfactory neurons, the primary olfactory epithelium can be dissected from the mouse before the neurons are dissociated. The dissected olfactory epithelium can be transferred to a dissociation buffer for mechanical and enzymatic dissociation. The dissociated neurons are then seeded on a cover slip to allow screening of thousands of cells by fluorescence microscopy, and a calcium-sensitive dye (Fura-2 AM) can be loaded onto the cells, for example, for about 30 min at 31 °C, which can then be transferred to the microscope ready for screening. The cells are stimulated by perfusing the dissociated olfactory neurons with a dilute solution of odorant (in saline). The rare cells that respond to odorous compounds are stimulated by stimulating the receptors with, for example, 50 μm odorous compound, and then detecting intracellular Ca as indicated by a change in Fura-2 fluorescence. 2+ The odorant receptor genes expressed as mRNA in the responsive cells are identified by monitoring the flux. After analysis, the responsive cells can be retrieved from the glass coverslip with a suction micropipette. The isolated cells are then pooled into one sample or processed individually for subsequent identification of the odorant receptor genes expressed as mRNA in the responsive cells.

[0072] In certain embodiments, olfactory neuron mRNA is purified and amplified according to the methods generally described in Marko, NF 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) technology or hybridized to known genes using microarray technology. NGS is generally described and described in Metzker, ML Nat. Rev. Genet. 11, 31-46 (2010). In certain embodiments, a minimum of five neurons exhibiting the same response profile are pooled. The mRNA is released by cell lysis immediately after harvesting; no DNAse and purification steps are performed. The mRNA is amplified by two successive rounds of in vitro transcription (IVT). Amplification may be performed according to the MessageAmpII aRNA kit (Ambion, AMA1751) with the following parameters: two consecutive 14-hour IVTs.

[0073] In a further embodiment, the mRNA of a single olfactory neuron is purified and amplified by a 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 then amplified with 18 PCR cycles to serve as the NGS sample for transcriptome sequencing.

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

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

[0076] Human or non-human mammalian DMTS receptors can be adapted to functional assays that can be used to identify compounds that bind, suppress, block, inhibit, and / or modulate the activity of olfactory receptors.In particular, the assays can be cell-based assays or binding assays, and the methods for identifying compounds can be high-throughput screening assays.More specifically, the present specification provides receptor-based assays that can be adapted to high-throughput screening of receptors with compound libraries for the discovery of regulatory compounds (e.g., binding, blocking, inhibiting, suppressing, and masking).

[0077] In a particular embodiment, DMTS receptor gene sequences are identified from DMTS-sensitive cells as follows: pooled neurons are heated to 75 °C for 10 min to disrupt cell membranes and make their mRNA available for amplification. This amplification step is important when applying NGS techniques with limited amounts of starting material (usually 1-15 cells). Linear amplification by the Eberwine method (IVT) ensures the maintenance of relative transcription levels of expressed genes. Sufficient amounts of cRNA are obtained using two consecutive nights (14 h) of in vitro transcription; the amplified cRNA is then used to generate an Illumina HiSeq cDNA library. The resulting short sequences (commonly called "reads"), usually 75-150 base pairs, are aligned against a 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 transcribed odorant receptor genes and their respective expression levels. The odorant receptor gene that shows the most abundant levels of mRNA (the most abundant "read") or is present in multiple replicate experiments is considered the putative DMTS receptor.

[0078] The predicted mouse OR genes are then used to mine the latest version genome databases of both mice and humans to identify the most closely related receptors (i.e., highest sequence similarity) in mice (paralogous genes) and humans (orthologous genes). This process can be performed using the sequence similarity search tool BLAST search algorithm (published on the NCBI website), where all 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 to be potential DMTS receptors under the assumption that paralogous and orthologous genes are likely to have similar activity. In certain embodiments, pairwise sequence homology comparisons are performed to identify closely related receptors in mice and humans, and receptors are identified as described in WO 2014 / 210585. Other approaches such as RT-PCR and microarray approaches can also be used.

[0079] In a further embodiment, to complete the deorphanization process, the candidate OR genes are further expressed in vitro to confirm activity against compounds used to isolate olfactory neurons and other structurally related compounds of interest. Mouse receptors identified from isolated olfactory neurons that respond to DMTS are modified at their N-terminus with short polypeptide sequences (e.g., FLAG® (SEQ ID NO: 44), Rho (SEQ ID NO: 46; first 20 amino acids of bovine rhodopsin receptor), and / or Lucy (SEQ ID NO: 48; cleavable leucine-rich signal peptide sequence) tags), transiently expressed in HEK 293T cells, and stimulated individually 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 of Gα activates the Gs transduction pathway, which leads to an increase in internal cAMP upon binding to an appropriate ligand. Alternatively, co-expression of the human G alpha subunit Gα in cell-based assays 15 Co-expression of Ca2+ induces internal Ca2+ expression upon binding to the appropriate ligand. 2+ The process described above and the results obtained so far serve to validate a process for rapid and reliable identification of mammalian odorant receptors for DMTS or DMDS.

[0080] Further, an assay is provided for identifying compounds that bind to DMTS or DMDS odorant receptors.In a further embodiment herein, there is provided a malodor regulating compound that binds, inhibits, blocks, inhibits and / or modulates the activity of at least one olfactory receptor selected from the group consisting of Olfr1193, Olfr1093, Olfr1097, Olfr166, OR52N5, OR2L13, OR4C15, OR5AC2, OR8H3, OR11G2 and OR52N2, and is identified by the method described herein, such as the compound described in Figure 4.

[0081] In one embodiment, the activity of the compound is determined by comparing its binding to that of DMTS or DMDS. In another embodiment, the receptor or chimera 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 chimera or fragment thereof that is activated by DMTS or DMDS, where the receptor or chimera or fragment thereof is expressed in a cell that has been recombinantly modified to express the receptor polypeptide.

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

[0084] In another embodiment, the contacting is with a liposome or a virus-induced budding membrane comprising a polypeptide described herein.

[0085] In another embodiment, the method for identifying compounds that bind, inhibit, block, inhibit and / or modulate the activity of olfactory receptors that can be activated by DMTS or DMDS can be performed on intact cells or membrane fractions from cells expressing the polypeptides described herein, or on olfactory neurons in culture that have been modified to express endogenous or exogenous odorant receptors.

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

[0087] The nucleic acid and amino acid sequences identified and / or used herein are listed below: Olfr1193 DNA-SEQ ID NO:1 atggcctcaaggacttattccatggaaagtaataataatgtcactgaattcattttcttgggtctttctcagaaccctgaggttgaaaagtgtgctttgtggtgttctccttctttacatggtcattctgctaggaaacctcctcatcatgttgacagtttgcagtggcaatcttttcaagtttcccatgtatttttcctcaactttctgtcttttgtggacatttgctactcct agtcacagcacccaagatgattattgacctgttagtgaagaaaaagactatatcctatgtggggtgcatgttacaactctttgtggttcatttctttggttgcactgagatcttcattcttactgtcatggcctatgatagatatgtggccatttgtaaacctctccactatatgactatgatggaccgggaaaagatgcaataagatgttgctcggaacatggatcggtggcttcttac attctattatccaagtggctcttgtggtccagctccccttttgtggaccgaatgagattgatcactatttctgtgatgtacatcctgtactgaaacttgcctgcactgacacttacattgttggtatttttgtgacagcaaacagtggcaccattgcattgggaagtttgtcatcttgctgatctcatacacagtcattcatgtctctgagaaagcagtcatctgaaggcagacg aaagctctctccacttgtggatcccacattgctgttgtcatcatttttttggcccctgtacttttatgtatatgcggcctgacactaccttctctgaggacaagatggtagctatattttacaccattatcactcccatgctgaatcctctaatttacactctaagaaatgcagaagtaaagaatgcaatgagaaaactgtgggctagaaagtttcctgggaaactactgggaaatag protein-sequence number 2 MASRTYSMEEVNNVTEFIFLGLSQNPEVEKVCFVVFSFFYMVILLGNLLIMLTVCSGNLFKFPMYFFLNFLSFVDICYSSVTAPKMIIDLLVKKKTISYVGCMLQLFVVHFFGCTEIFILTVMAYDRYVAICKPLHYMTMMDRERCNKMLLGTWIGGFL HSIIQVALVVQLPFCGPNEIDHYFCDVHPVLKLACTDTYIVGIFVTANSGTIALGSFVILLISYTVILMSLRKQSSEGRRKALSTCGSHIAVVIIFFGPCTFMYMRPDTTFSEDKMVAIFYTIITPMLNPLIYTLRNAEVKNAMRKLWARKFSWETTGK Olfr1093 DNA-SEQ ID NO:3 atggaaaagatcacatcagctgtggatgtccacaatattccattaaagaaacatgactgaagccaccatgtttattctcttaggattcacagatgactttgaactccaagtcttcctgtttttactgtttcttgctatttatctcttcactctggtaggaaactttggactggttgttttggtcattggggattgtcggctacacaaccccatgtactatttcctaagtgttttgtctttcct ggatgcttgctattctacagttgttacacccaaaatgttggtcaactttctaagtgaaaataagtccatttcattccttgcatgtgcaacccaaatgcttctctttgtttcgttgggaaccacagaatgctttctcctggcagcaatggcttatgaccgatatgtagccatctacaacccacttctgttatacagtggccatgtcacccagagtatacctgccactcatcattgcttcct ctggtggagttgtgcatggtgctatccacacagtggccactttcagtctgtccttctgtggatccaatgaaattaagcatgtcttctgtgacatccctgcattgcttgctctttcttgttctgatacccacaacaaatgagcttctagtcttgtacttggtgggcttgattgagattgttaccatcctgattgttctggtctccttggattcatcctctttgccattctgaacatgcattct gctgagggtaggaggaaagtgttctctacatgtggctctcacctcactggagtctctatttaccatggtacaatccttttcacttatatgaggcctagttccagttatgcttcaaatcatgacatggtagtgtcaatattttacaccattgtgatacccatgttgaatcctatcatctatagtttgaggaacaaagatgtaaaagtagcatttaataaattgtggagaaaatgtgattcataa protein-sequence number 4 MEKITSAVDVHNIPLKNMTEATMFILLGFTDDFELQVFLFLLFLAIYLFTLVGNFGLVVLVIGDCRLHNPMYYFLSVLSFLDACYSTVVTPKMLVNFLSENKSISFLACATQMLLFVSLGTTCFLLAAMAYDRYVAIYNPLLYTVAMSPRVYLPLIIASY AGGVVHGAIHTVATFSLFCGSNEIKHVFCDIPALLALSCSDTHTNELLVLYLVGLIEIVTILIVLVSYGFILFAILNMHSAEGRRKVFSTCGSHLTGVSIYHGTILFTYMRPSSSYASNHDMVVSIFYTIVIPMLNPIIYSLRNKDVKVAFNKLWRKCDS Olfr1097 DNA sequence number 5 atgagtgcctgtaatcatacaaatgaacctgagttcacgcttgtgggactgacagactccaagggattcagctggtcctctctgttttgtttctcctgatatacatgctcactgtcttgggaaaacataggtatgatactgatcattcatctagatgtccagctccacactccaatgtatttttcctcaccacttgtcattccttgacctcagttactcaactgtaatcacacct aaaaccttacagaatacgctgacctccataaaaaatatttccttcatgggatgcttcacccagttgtatttctttgtcctcttggcagcttctgaatgttttatactttcgtcaatggcctatgaccgctatgtagctatctgcaaccctctacactatccagttattatgtcccctaggcgctcatatactctcatcactgtgtcctacatgattggagttttggattcttctgtc actgtcttttgcttaagcacactggatttctgcaactccaaagtaattcatcacttttttgtgacacattcccaattttagctctgtcctgcagtgatacctataatgcagaagccactatattcgttttaggctggttccactctattgctgtcgctcatcacgatatcctcatcctatgtatctattctctctacaattttgaagaataaattcttcttcaggaaagcacaaaagcc ttctctacatgtgcctcacatcttataggagtcactgttttttatggtacaatgatctttacttatttaaaaaccaagtacgtcctactccctgggaaaggatcaagtagcctctgttttttatactatagtgattcccatgctgaacccacttatctatagtctcaggaacaaagaagtgaaagtgctgttgttaggttatgaagaagagagagtgcatccagaaactagaataa protein-sequence number 6 MSACNHTNEPEFTLVGLTDSKEIQLVLSVLFLLIYMLTVLGNIGMILIIHLDVQLHTPMYFFLTHLSFLDLSYSTVITPKTLQNTLTSIKNISFMGCFTQLYFFVLLAASECFILSSMAYDRYVAICNPLHYPVIMSPRRSYTLITVSYMIGVLDSSVTVFCLSTLDFCNSKVIHHFFCDTFPILALSCSDTYNAEATIFVLAGSTLLLLSLITISSSYVSILSTILKINSSGKHKAFSTCASHLIGVTVFYGTMIFTYLKPSTSYSLGKDQVASVFYTIVIPMLNPLIYSLRNKEVKSAVVRVMKKRECIQKLE Olfr166 DNA sequence number 7 atggagaaatggaatcagagctcaagtgattttactctgttaggactgcttccacaaaaccaaacaggcctgctacttttgatgctcatcatctttgtcttctctctggctttgtgtggcaactcaggaatgatccacctcattcgtgtggatccaaggctccacacccccatgtactttctcctcagtcagctctctctcatggacctgatgtacatttctaccactgttccc aagatggcatttaacttccttctggccagaaaagcatctcttttctgggctgtggagtgcaatccttttcttcctgactatggcatgttctgagggcttgctcttggcttccatggcttatgatcgttttgtggctatctgccatccccttcactatcccattcgcatgagcaaaaataatgtgtctgaagatgatcataggatcctggatattgggctcaatcaactctttag cacataccgtctatgcccttcatattccttactgccattctaggtccattaaccatttttctgtgatgttccagccatgttgcccctggcctgtatggacacttgggtttatgagtacatggtgtttgtgagcacaagcctgtttctcctactgcctttccttggtatcacagcttcctatggtcgggtcctttttgctgtcttcacatgcgctcaaaagagggaaaagaagaa ggccttcaccacatgctcaactcacttaactgtggtgacatttactatgcaccttttgtctatacctatcttcgacctaggagtcttcgctccccaacacaagagataagattctggctgtttctacactatccttacccccatgctcaaccccatcatttatagtctgaggaataaggaggtcctgggggccatgacaagagtccttggtacttttccttcaactaaaccgtaa protein-sequence number8 MEKWNQSSSDFTLLGLLPQNQTGLLLLMLIIFVFSLALCGNSGMIHLIRVDPRLHTPMYFLLSQLSLMDLMYISTTVPKMAFNFLSGQKSISFLGCGVQSFFFLTMACSEGLLLASMAYDRFVAICHPLHYPIRMSKIMCLKMIIGSWILGSINSL AHTVYALHIPYCHSRSINHFFCDVPAMLPLACMDTWVYEYMVFVSTSLFLLLPFLGITASYGRVLFAVFHMRSKEGKKKAFTTCSTHLTVVTFYYAPFVYTYLRPRSLRSPTEDKILAVFYTILTPMLNPIIYSLRNKEVLGAMTRVLGTFPSTKP Olfr169 DNA-SEQ ID NO:9 atggaatatgagaactacacttttaacagcgacttcatcctcttgggactgttctcttcttcaaaagacaagcttaactttttctcatttatatttttcatttttattatggctataacagaaaatgccctcatgatcctcctaatccacagggattctcgactccataccccaatgtatttcctgcttagtcatctctccttcatggatatcttgcacatttccaacattgttc ctaaaatgattgctgacttctctcaggcagcagaactatttcctttgcaggctgtgccttccagatattctctctcttaccttgctaggtggtgagtgccttctcctggcagccatgtcctatgatcgatatgtggccatctgccaccacttcctaccctgtgctgatgagggataactccagtaggctcctggctccaggctcctggctggtggggatcctcaactccata gtacacacagtttttgcactccactttcccttctgccactcaagagccattgatcacttttctgcgaagtccctgccatgttgaaattgtcatgtatagacacaacactatgaacgaggcgtttatgtgagtggcattatttttctgctgatcccattttccatgatctctatatcttatgtgcaaattctcctcactgtattccaaatgcagtcatgggcccggcaaacag agtccttttccacctgttccttccacatggttgttgtcataatgtactatgggccattcatttttacatatatgagacctcgctcataccacactccagggcaggataaatttttggcaatattctacacatcctgacacccacactcaaccccataatctacagctttcgtaataaaagatgtccttatggctgtgaaaaacatcgtccaaaagtaatttttgaataaaaaatga protein-sequence number 10 MEYENYTFNSDFILLGLFSSSKTSLTFFSFIFFIFIMAITENALMILLIHRDSRLHTPMYFLLSHLSFMDILHISNIVPKMIADFLSGSRTISFAGCAFQIFLSLTLLGGECLLLAAMSYDRYVAICHPLRYPVLMRDNSSRLLAAGSWLVGILNS IVHTVFALHFPFCHSRAIDHFFCEVPAMLKLSCIDTTHYERGVYVSGIIFLLIPFSMISISYVQILLTVFQMQSSGARQKSFSTCSFHMVVVIMYYGPFIFTYMRPRSYHTPGQDKFLAIFYTILTPTLNPIIYSFRNKDVLMAVKNIVQSNFLNKK Olfr738 DNA-SEQ ID NO:11 atgaaagcctttagcagccccagcaactccagcatcatcactggcttcatcctcctgggcttccctgccccaaggaggggcaaatcctcctctttgtgctcttcttcattatctacatccttaccctcatgggcaatgcttccatcatatgtgctgtgtgctatgataagaaacttcacagccccatgtacctcctgctggccaacttctcttcctagaaatctggtatgtc acctccacagtccccaaacatgttggccaacttctctctgacacgaaggtcatctctttctctggatgcttcctgcagttctatttcttcttctccttgggttctacagaatgctttttcctggcagtcatggcatttgatcgataccttgccatctgcagacctcatattatccttctctcatgactgggcgctctgcaacatccttgtgatcagttgctgggtgcttggt ttcctctggttccctgttcccatcatcatcatctcccaaatgtccttctgtggatccagaattatagaccttcctgtgtgacccaggccctctgttggccctcacctgtgtgagaaattctttaattgagatgactagctctactttaagtccctgcttttatttgttccattttttttatcatggggtcttatgctctagtaatgagggctgtgctcagggtcccttca gcagctggacgaagaaaggccttctccacctgtgggtcacacttgactgtggtttctcttttctatggctcagtgatggtcatgtatgtgagcccaacatctgaacatgcagctggagatgcaaaaacttgtgactctgttttattctgtggttactcccctccttaatcctgtgatatacagtctgaggaacagagatatgaaacatgcaatgaaaaagttactgaaaatgtaa protein-sequence number 12 MKAFSSPSNSSIITGFILLGFPCPKEGQILLFVLFFIIYILTLMGNASIICAVCYDKKLHSPMYLLLANFSFLEIWYVTSTVPNMLANFLSDTKVISFSGCFLQFYFFFSLGSTECFFLAVMAFDRYLAICRPLHYPSLMTGRLCNILVISCWVLGFLWFPVPIIIISQMSFCGSRIIDHFLCDPGPLLALTCVRNSLIEMTSSTLSSLLLFVPFFFIMGSYALVMRAVLRVPSAAGRRKAFSTCGSHLTVVSLFYGSVMVMYVSPTSEHAAGVQKLVTLFYSVVTPLLNPVIYSLRNRDMKHAMKKLLKM Olfr742 DNA sequence number 13 Atgaaaaccctcagcagccccagcaactccagcaccatcactggcttcatcctcttgggcttccctgccccagggaggggcaaatcctctctttgtgaccttcttcattgtttacatactcattcttatgggcaatgcttccatcatctgtgctgtgtactgtgatcagagcctccacacccccatgtacttcctgctggccaacttctcttcctggagatctggtatgtt acctccacagtccccaacatgttggccaacttcctttcagacaccaaggtcatctctttctctggatgcttcctgcagttctatttcttcttctcctttggttctacagaatgctttttcctggcagtcatggcatttgatcgataccttgccatctgtaggccactacattatccttctctcatgactgggcacctctgcaacatccttgtgatcagttgctggtgcttggt ttcctctggtccctgtacccatcatcatcatctcccagatgtccttctgtgggtccagaattatagaccttcctgtgtgacccaggccctcttttggcccttgcctgttccagagccccattgatggaggttttctggacaattataatgtctatgctcctggttattccttcctcttcatcatgggaacttacatattggtcctaagagcttgtgtttagacttcttca agagatggacaaaaaaggccttctccacttgcgggtctcatctcacagtagtttcactctttattgctcagtgatgaaaatgtatttgagcccaacatctgagcatgaagctggaatgcagaagcttgtaactctatttattctgtgggtactccactacttaatcctgtgatatacagtctgaggaacaaaagatatgaaaaatgccctgcagaagattttaagaacataa protein-sequence number 14 MKTLSSPSNSSTITGFILLGFPCPREGQILLFVTFFIVYILILMGNASIICAVYCDQSLHTPMYFLLANFSFLEIWYVTSTVPNMLANFLSDTKVISFSGCFLQFYFFFSFGSTECFFLAVMAFDRYLAICRPLHYPSLMTGHLCNILVISCWVL GFLWFPVPIIIISQMSFCGSRIIDHFLCDPGPLLALACSRAPLMEVFWTIIMSMLLVIPFLFIMGTYILVLRAVFRLPSRDGQKKAFSTCGSHLTVVSLFYCSVMKMYLSPTSEHEAGMQKLVTLFYSVGTPLLNPVIYSLRNKDMKNALQKILRT Olfr207 DNA-SEQ ID NO:15 atggaactgaacaggacccagctgactgaatttgttctcagaggaataacagatcgttcagagctgcaagtccccctgttcctggtgttctttctcatctatgttatcaccatggtgggcaaccttggcttaatctttgtcatctggaaggaccctcatcttcacacacccatgtaccttttccttggaaatttggcctttgctgatgcctgtaattcatcctctgtga caccaaagatgcttatgaaatttttaaataagaatgacatgatatccatgggtgagtgttttgctcaattttatttcttttgttcaagtgtaactgcagaagccttcattctggtagctatggcctatgaccgctatgtagccatatgcaaacctctgctctatgtagtggtgatgtccaacagactctgtattcagttcataggtgtatcctatctaattggacttcta catggcttacttcatgtaggattgttatttaggttaacgttttgtagttccaatgtaatagatatttctactgtgacatcctgccactttataggatttcttgcactgacccatcgatcaatgtactggtagctttcattatgggtattttattacaagtgagtacctttatgagtattatagtctcctatgtccgtgtcctctttgccatcctgagaacaaagtctg agaggggcagaaacaaagccttctctacttgcagttcccacctgtcatctgtgtctttgttctatggcactctcttcatcatatatgtcctctctggctctgacacagataattatcagggtaaatgtattcactgttctataccattatcattcctctgctaaaccccttcatttacagcctaagaaataagaagtcatcggtgccttgagaaaagtcagaaaatga protein-sequence number 16 MELNRTQLTEFVLRGITDRSELQVPLFLVFFLIYVITMVGNLGLIFVIWKDPHLHTPMYLFLGNLAFADACNSSSVTPKMLMKFLNKNDMISMGECFAQFYFFCSSVTAEAFILVAMAYDRYVAICKPLLYVVVMSNRLCIQFIGVSYLIGLLHGLLHVGLLFRLTFCSSNVIDYFYCDILPLYRISCTDPSINVLVAFIMGILLQVSTFMSIIVSYVRVLFAILRTKSERGRNKAFSTCSSHLSSVSLFYGTLFIIYVLSGSDTDNYQGKMYSLFYTIIIPLLNPFIYSLRNKEVIGALRKVRK Olfr665 DNA sequence number 17 atgcctggggtcaatacctccagcctgacaccaagatactttatctcaatgggattcctgggttggaagctgcacacatctggatctctctgccattcttcattgtacctcattgctgtcacaggtaactgtggacttatctacctcatcagtcatgaggagctctgcaccggcccatgtactactttctagccatgttgtctgctacagatatttctgggtgtaatacaatt gtccccagtatgttatgcatcttttggttcagtgtcaaggagattgatttcaatgcctgccttgtacagatgtttttcatccacatgttaacaggcatggagtctggtgtgctcatgcttatggctctcgaccgctatgtggctatatgctatccattacgttatactaccatactcaccaacactatgattaccaagattggattggcagcacttgttagaagtgtgttactcatgg tcccttttgctttcctgatcaagcgtcttccacatactgtagaggaaacctcatccaacacatacctattgtgatcacatgggcttggctaaactatcctgtggcaatattaagattaatgctatctatggtcttataattgctatatttattgggggttttgatatattctgtatctccatgtcttatgccatgattatccatgctgtggtgaagctatcttcggcagatgctcgccataa agccttcagtactgtgacatcacacatatgtgctattgttatactactatgtcccagcattcttcaacttctttactcatcgctttgggagaacactataccccatcatatccacattattatagccaacctgtatctattgctacctcccaccttgaatccaattgtatatggagtaaaagaccaagcagattcgtgaaggtgtgatcaaactgtttgctagaaaaagttgtttga protein-sequence number 18 MPGVNTSSLTPRYFILNGIPGLEAAHIWISLPFFIMYLIAVTGNCGLIYLISHEEALHRPMYYFLAMLSATDISGCNTIVPSMLCIFWFSVKEIDFNACLVQMFFIHMLTGMESGVLMLMALDRYVAICYPLRYTTILTNTMITKIGLAALVRSVLLM VPFAFLIKRLPYCRGNLIQHTYCDHMAVAKLSCGNIKINAIYGLIIAIFIGGFDIFCISMSYAMIIHAVVKLSSADARHKAFSTCTSHICAIVITYVPAFFNFFTHRFGRTTIPHHIHIIIANLYLLLPPTLNPIVYGVKTKQIREGVIKLFARQKVV Olfr669 DNA-SEQ ID NO:19 atgctgatttccaacaactcatatgaagccccgcagtctttcattcttaatggaattcctggtctcgaagcagtgcatatatggatctctcttccactctgtacaatgtacatcatctccctagtaggcaaccttggccttgtatatctcatttactatgaggaatccttacatcgcccaatgtatttctttctggccatgctttctctcatagacctgtttacttgcacaaccactg tccccaatgccctcttcatttctggttcaaactcaaggaaattaacttcactgcttgcctagttcagatgttctttgtgcacggattcacaggtgtggagtctggggtactcatgctcatggccttggaccgctatgtggccatttgctacccactacgctatgcaaccatacttaccaaccctgtcattgccaaagctggcttgccaccttcttgagaggtgtgttactgatgatt ccttttccattcttggttaaacgtttgcccttctgccgaagcaatgtcatctcccatacatattgtgaccacatgtctgtggtaaagttatcctgtgccagcatcaaaatcaatgtcatctatggtctcatggttgcacttctgattggagtgtttgacatatgttgtatatctgtgtcctacactatgatcctccgggcagtggtcagcctgtcctctgcagatgctcggcagaagg ccttcagcacctgcacagcccacatatctgccatcatcattacttatgttccagccttcttcaccttttactcatcgttttggaggtcacaccatccctccttctcttcatatcatgtggctaatctttatcttcttctccctccaactctaaatcccattgtttatgggatgaagaccaaacagatcagagatagtatcattaaattctttcacggtgaaaaaggttcaaggtga protein-sequence number 20 MLISNNSYEAPQSFILNGIPGLEAVHIWISLPLCTMYIISLVGNLGLVYLIYYEESLHRPMYFFLAMLSLIDLFTCTTTVPNALFIFWFKLKEINFTACLVQMFFVHGFTGVESGVLMLMALDRYVAICYPLRYATILTNPVIAKAGLATFLRGVLLM IPFPFLVKRLPFCRSNVISHTYCDHMSVVKLSCASIKINVIYGLMVALLIGVFDICCISVSYTMILRAVVSLSSADARQKAFSTCTAHISAIIITYVPAFFTFFTHRFGGHTIPPSLHIIVANLYLLLPPTLNPIVYGMKTKQIRDSIIKFFHGEKGSR Olfr1211 DNA-SEQ ID NO:21 atgcaaaccagagtttgtaacagaattcatattccttggactttcacagaaccctaaagtccagaaaatagttttattgttattttatttgtctacattgcaactgttggggcaacatgataattgtggtgaccattgtctgtagcccagcattgatagactgccccatgtacttctttttggcattcttgtccctattggatgcatgcttctcttctgtcatcacacca aagatggttgtggactccctgttgagaagaaaactatctcctttgaaggagttatgatgcagttatttgctgagcacttccttgcagcagtagaagtgattgtcttgacagccatggcctatgaccgctatgtagcaatttgcaagcccttgcactactcttccatcatgaactggaggctctgtggcacacttatggggatagcatggacaggggggctttgcattctatc atacaaattatcttcacgttgcaattgcccttctgtggaccaaatgtcatcgatcatttcatgtgtgacttgttcccattactggaacttgcctgcactgatactcatatctttggccttttagtggttgccaacagtgggtctatctgcatcataatcttctctattttgctggtctccttggtgtcatcctgttctctctgaaagctcacagttctgaagggcgatggaaa gctctctccacatgtggatcccacattgcagttgtggttttgttctttgtcccgtgtatatttatttatgcacgtcctccatctgctttctcctttgataaaatggtggcgatatttatactatcctaactcccttgctcaatcctgatttaacttttcggaataaggacatgaaaaatgctatgaagaaagtgtggagaggttggcagtggtttctgatggaaagtga protein-sequence number 22 MQNQSFVTEFIFLGLSQNPKVQKIVFIVFLFVYIATVGGNMIIVVTIVCSPALIDCPMYFFLAFLSLLDACFSSVITPKMVVDSLYEKKTISFEGCMMQLFAEHFLAAVEVIVLTAMAYDRYVAICKPLHYSSIMNWRLCGTLMGIAWTGGFLHS IIQIIFTLQLPFCGPNVIDHFMCDLFPLLELACTDTHIFGLLVVANSGSICIIIFSILLVSYGVILFSLKAHSSEGRWKALSTCGSHIAVVVLFFVPCIFIYARRPPSAFSFDKMVAIFYTILTPLLNPVIYTFRNKDMKNAMKKVWKRLAVVSDGK OR52N5 DNA-SEQ ID NO:23 atgcctctatttaattcattatgctggtttccaacaattcatgtgactcctccatcttttattcttaatggaatacctggtctggaaagagtacatgtatggatctccctcccatctgcacaatgtacatcatcttccttgtggggaatcttggtcttgtgtacctcatttattatgaggatccttacatcatccgatgtatttttttttggccatgctctctccctcattgacctcctt acctgcaccaccactctacccaatgcactctgcatcttctggttcagtctcaaagaaattaacttcaatgcttgcttggcccagatgttctttgttcatgggttcacaggtgtggagtctggggtgctcatgctcatggctctagaccgctatgtagccatttgctaccctttgcgttatgctaccacatcaccaaccctatcattgccaaggctgagcttgccaccttcctgagggtgtat tgctgatgattcctttcccattcttggttaagcgtttgcctttctgccaaagcaatattatctcccatacgtactgcgaccacatgtctgtagtaaagctatcttgtgccagcatcaaggtcaatgtaatctatggtctaatggttgctctcctgattggagtgtttgacatttgttgtatatctttgtcttacactttgatcctcaaggcagcgatcagcctctcttcatcagatgctcggca gaaggctttcagcacctgcactgcccatatatctgccatcatcatcacctatgttccagcattcttcactttctttgcccaccgttttggggcacacaattcccccttctcttcacatcattgtggctaatctttatcttcttcttcccccaactctaaaccctattgtttatggagtaaagacaaaacagatacgcaagagtgtcataaagttcttccagggtgataagggtgcaggttga protein-sequence number 24 MPLFNSLCCWFPTIHVTPPSFILNGIPGLERVHVWISLPLCTMYIIFLVGNLGLVYLIYYEESLHHPMYFFFGHALSLIDLLTCTTTLPNALCIFWFSLKEINFNACLAQMFFVHGFTGVESGVLMLMALDRYVAICYPLRYATTLTNPIIAKAELATFLRGV LLMIPFPFLVKRLPFCQSNIISHTYCDHMSVVKLSCASIKVNVIYGLMVALLIGVFDICCISLSYTLILKAAISLSSSDARQKAFSTCTAHISAIIITYVPAFFTFFAHRFGGHTIPPSLHIIVANLYLLLPTLNPIVYGVKTKQIRKSVIKFFQGDKGAG OR2L13 DNA-SEQ ID NO:25 atggagaaatggaatcacattcaaatgatttcattttgttgggtctgcttcccccaaatcaaactggaatatttctcttgtgccttatcatcctcatattctttctggcctcggtgggttaactcggccatgattcacctcatccacgtggatcctcgtctccacacaccgatgtactttcttctcagccagctctccctttggacctgatgtacatctccaccaccgtcccc aagatggcgtacaacttcctgtccggccagaaaggcatctccttcctgggatgtggtgtgcaaagcttcttcttcctgaccatggcgtgttctgaaggcttactcctgacctccatggcctacgaccgttatttggccatctgccactctctctattatcctatccgcatgagtaaaatgatgtgtgtgaagatgattggaggctcttggacactgggtccatcaactccttgg cacacacagtctttgcccttcatattccctactgcaggtctagggctattgaccatttcttctgcgatgtcccagccatgttgcttcttgcctgtacagatacttgggtctatgaatatatggtttttgtaagtacaagcctctttctcctttccctttcatggcatcacttcttcctgtggccgagtcctatttgctgtctatcatatgcactcaaaggagggaaaaaaa ggccttcaccaccatttcaacacatttaactgtagtgatcttttactatgcaccttttgtctacacctatcttcggcccaggaatctccgctcaccagctgaagacaagatcctggcagtcttctacaccatccttacccccatgctcaatcccattatctacagcctgaggaataaggaagtcctggggctatgaggagagtgtttgggatatctctttccgaaagaataa protein-sequence number 26 MEKWNHTSNDFILLLGLPPNQTGIFLLCLIILIFFLASVGNSAMIHLIHVDPRLHTPMYFLLSQLSLMDLMYISTTVPKMAYNFLSGQKGISFLGCGVQSFFFLTMACSEGLLLTSMAYDRYLAICHSLYYPIRMSKMMCVKMIGGSWTLGSINSL AHTVFALHIPYCRSRAIDHFCDVPAMLLLACTDTWVYEYMVFVSTSLFLLFPFIGITSSCGRVLFAVYHMHSKEGRKKAFTISTHLTVVIFYYAPFVYTYLRPRNLRSPAEDKILAVFYTILTPMLNPIIYSLRNKEVLGAMRRVFGIFSFLKE OR2AJ1 DNA sequence number 27 atgagtgtaacagaaaatacgctcatgatcctcccattcgcagtgactcccgactccacactccaatgtattttctgctcagccatctctccttaatggatatcttgcatgtttccaacatcgttcccaaaatggtcactaactttctgtcaggcagcagaactatttcatttgcaggttgtgggttccaggtatttctgtccctcaccctcctggt ggtgagtgccttctcctggctgcaatgtcctgtgatcgctatgtggctatctgtcacccgctgcgctatccgattcttatgaaggagtatgccagcgctctcatggctggaggctcctggctcattggggttttcaactccacagtccacacagcttatgcactgcagtttcccttctgtggctctagggcaattgatcacttcttctgtgaagtcccttg ccatgttgaagttgtcctgtgcagacacaacacgctatgaacgaggggtttgtgtaagtgctgtgatcttcctgctgatccctttctccttgatctctgcttctttatggccaaattatcttactgtcctccagatgaaatcatcagaggcaaggaaaaagtcattttccacttgttccttccacatgattgtggtcacgatgtactatgggccatttat ttttacatatatgagacctaaatcataccacactccagggcaggataagttcctggcaatattctatacgatcctcacacccacactcaaccctttcatctacagctttaggaataaagatgttctggcggtgatgaaaaatatgctcaaaagtaactttctgcacaaaaaaaatgaataggaaaattcctgaatgtgtgttctgtctatttctatgttaa protein-sequence number 28 MSVTENTLMILLIRSDSRLHTPMYFLLSHLSLMDILHVSNIVPKMVTNFLSGSRTISFAGCGFQVFLSLTLLGGECLLLAAMSCDRYVAICHPLRYPILMKEYASALMAGGSWLIGVFNSTVHTAYALQFPFCGSRAIDHFFCEVP AMLKLSCADTTRYERGVCVSAVIFLLIPFSLISASYGQIILTVLQMKSSEARKKSFSTCSFHMIVVTMYYGPFIFTYMRPKSYHTPGQDKFLAIFYTILTPTLNPFIYSFRNKDVLAVMKNMLKSNFLHKKMNRKIPECVFCLFLC OR4C15 DNA-SEQ ID NO:29 Protein - SEQ ID NO:30 MFSMTTEALNNFALGCTNLLMTMIPQIDLKQIFLCPNCRLYMIPVGAFIFSLGNMQNQSFVTEFVLLGLSQNPNVQEIVFVVFLFVYIATVGGNMLIVVTILSSSPALLVSPMYFFLGFLSFLDACFSSVITPKMIVDSLYVTKTISFEGCMMQLFAEHFFAGVEVIVLTAMAYDRYVAICKPLHY SSIMNRRLCGILMGVAWTGGLLHSMIQILFTFQLPFCGPNVINHFMCDLYPLLELACTDTHIFGLMVVINSGFICIINFSLLLVSYAVILLSLRTHSSEGRWKALSTCGSHIAVVILFFVPCIFVYTRPPSAFSLDKMAAIFYIILNPLLNPLIYTFRNKEVKQAMRRIWNRLMVVSDEKENIKL OR5AC2 DNA-SEQ ID NO:31 atggatatatcagagggaaataagactcttgtgacagagtttgttctcacaggacttacagatcgaccatggctgcacgtcctcttctttgttgtgtttttggtggtctatctcatcaccatggtgggcaaccttggactgatagttctaatttggaacgacccccatcttcatatgcccatgtacttattccttggtggtttagccttttcagatgcttgtacttcaacct ctataacccctaggatgctggtcaatttcttagacaagactgcaatgatatccctaggctgagtgcatcacccagttttactttttgcttccagtgcaactacagaatgcttcctcctggtgatggcctatgaccgctatgtagccatatgtaatcccttgctttatccagtgatgatgtccaacaaactcagcgctcagttgctaagtatttcatatgtaattggtttc ctgcatcctctggttcatgtgagtttactattgcgactaactttctgcaggtttaacataatacattatttctactgtgaaattttacaactgttcaaaatttcatgcaatggtccatctattaacgcactatgatattatttttggtgcttttatacaaatacccactttaatgactatcataatctcttactcgtgtgctctttgatattctgaaaaaaaagtctg aaaagggcagaagcaaagccttctccacatgcggcgcccatctgctttctgtctcattgtactacggaactctgatcttcatgtatgtgcgtcctgcatctggcttagctgaagaccaagacaaagtgtattctctgttttacaccgattataattcccctgctaaacccatttatttacagcttgagaaataaaaaagtcatgcatgcattgaaagagttataaggaagtaa protein-sequence number 32 MDISEGNKTLVTEFVLTGLTDRPWLHVLFFVVFLVVYLITMVGNLGLIVLIWNDPHLHMPMYLFLGGLAFSDACTSTSITPRMLVNFLDKTAMISLAECITQFYFFASSATTECFLLVMMAYDRYVAICNPLLYPVMMSNKLSAQLLSISYVIG FLHPLVHVSLLLRLTFCRFNIIHYFYCEILQLFKISCNGPSINALMIFIFGAFIQIPTLMTIIISYTRVLFDILKKKSEKGRSKAFSTCGAHLLSVSLYYGTLIFMYVRPASGLAEDQDKVYSLFYTIIIPLLNPFIYSLRNKKVMHALRRVIRK OR8H3 DNA-SEQ ID NO:33 atgatgggtagaaggaatgacacaaatgtggctgacttcatccttacgggactgtcagactctgaagaggtccagatggctctgtttatgctatttctcctcatatacctaattactatgctgggaatgtggggatgctattgataatccgcctggacctccagcttcacatcccatgtatttttccttactcacctgtcatttattgacctcagttactcaactgtcgtc acacctaaaaccttagcgaacttactgacttccaactatatttccttcacgggctctctttgcccagatgttctgttttgtcttcttgggtactgctgaatgttatcttctctctcaatggcctatgatcgctatgcagcgatctgcagtcctctacactacacagttattatgcccaaaggctctgcctcgctctcatcactgggccttatgtgattggctttatggactcct ttgtcaatgtggtttccatgagcagattgcatttctgtgactcaaacataattcatcactttttctgtgacacttcccaattttagctctgtcctgcactgacacagacaacactgaaatgctgatatcattatcgctggttccaccctgatggtgtcccttatcacaatatctgcatcctatgtgtccattctctctaccatcctgaaaattaattccacttcaggaaagca gaaagctttctctacttgcgtctctcatctcttgggagtcaccatcttctatggaactatgattttacttacttaaagccaagaaagtcttattccttgggaagagatcaagtggctcctgtgttttatactattgtgattcccatgctgaatccactcatttatagtcttagaaacagagaagtgaaaaatgctctcattagagtcatgcagagaagacaggactccaggtag protein-sequence number 34 MMGRRNDTNVADFILTGLSDSEEVQMALFMLFLIYLITMLGNVGMLLIIRLDLQLHTPMYFFLTHLSFIDLSYSTVVTPKTLANLLTSNYISFTGCFAQMFCFVFLGTAECYLLSSMAYDRYAAICSPLHYTVIMKPRLCLALITGPYVIGFMDS FVNVVSMSRLHFCDSNIIHHFFCDTSPILALSCTDTDNTEMLIFIIAGSTLMVSLITISASYVSILSTILKINSTSGKQKAFSTCVSHLLGVTIFYGTMIFTYLKPRKSYSLGRDQVAPVFYTIVIPMLNPLIYSLRNREVKNALIRVMQRRQDSR OR11G2 DNA sequence number 35 atgaaaatcttcaacagccccagcaactccagcaccttcactggcttcatcctcctgggcttcccttgccccagggaggggcagatcctcctctttgtgctcttcactgttgtttacctcctgaccctcatgggcaatggttccatcatctgtgctgtgcactgggatcagagactccacgcccccatgtacatcctgctcgccaacttctcttcttggagatatgttatgtc acctccacagtccccagcatgctggccaacttcctctctgacaccaagatcatctcgttctctgggctgcttcctccagttctacttttttctctccttgggctctacagaatgctttttcctggcagttatggcatttgatcgataccttgccatctgtcggcctctacgctatccaaccattatgaccagacgtctctgtaccaatcttgtggtcaattgctgggtacttggt ttcatctggttcttgattcctatcgtcaacatctcccaaatgtccttctgtggatctaggattattgaccacttcctatgtgacccagctcctcttctaactctcacttgcaaaaaaggccctgtgatagagcttgtcttttctgtcttaagtcctctgcctgtctttatgctctttctcttcattgtggggtcctatgctctggtcgtgagagctgtgttgaggtcccttca gcagctgggaagaaaagggctttctccacctgtgggtctcacctggctgtggtttcactgttctacggctcagtactggtcatgtatgggagcccaccatctaagaatgaagctggaaagcagaagactgtgactctgttttattctgttgttaccccactgcttaaccctgtgatatatagtcttaggaacaaaagatatgagaaaagctctgaagaaattttgggaacataa protein-sequence number 36 MKIFNSPSNSSTFTGFILLGFPCPREGQILLFVLFTVVYLLTLMGNGSIICAVHWDQRLHAPMYILLANFSFLEICYVTSTVPSMLANFLSDTKIISFSGCFLQFYFFFSLGSTECFFLAVMAFDRYLAICRPLRYPTIMTRRLCTNLVVNCWVL GFIWFLIPIVNISQMSFCGSRIIDHFLCDPAPLLTLTCKKGPVIELVFSVLSPLPVFMLFLFIVGSYALVVRAVLRVPSAAGRRKAFSTCGSHLAVVSLFYGSVLVMYGSPPSKNEAGKQKTVTLFYSVVTPLLNPVIYSLRNKDMRKALKKFWGT OR52N2 DNA-SEQ ID NO:37 atgtctggggacaacagctccagcctgaccccaggattctttatcttgaatggcgttcctgggctggaagccacacacatctggatctccctgccattctgctttatgtacatcattgctgtcgtgggggaactgtgggctcatctgcctcatcagccatgaggagccctgcaccggcccatgtactacttcctggccctgctctcttcactgatcaccttgtgcaccaccatggtac ctaatatgctgtgcatattctggttcaacctcaagggattgactttaacgcctgcctggcccagatgttttttgtccatgctgacagggatggagtctggggtgctcatgctcatggccctggaccgctatgtggccatctgctaccccttacgctatgccaccatccttaccaaccctgtcatcgccaaggctggtcttgccaccttcttgaggaatgtgatgctcatcatcccattc actctcctcaccaagcgcctgccctattgccgggggaacttcatcccccacacctactgtgaccatatgtctgtggccaaggtatcctgtggcaatttcaaggtcaatgctatttatggtctgatggttgctctcctgattggtgtttgatatctgctgtatctctgtatcttacactatgattttgcaggctgttatgagcctgtcatcagcagatgctcgtcacaaagccttcagca cctgcacatctcacatgtgttccattgtgatcacctatgttgctgcttttttcacttttttcactcatcgttttgtaggacacaatatcccaaaccacatacacatcatcgtggccaacctttatctgctactgcctcctaccatgaacccaattgtttatggagtcaagaccaagcagattcaggaaggtgtaattaaatttttacttggagacaaggttagttttacctatgacaaatga protein-sequence number 38 MSGDNSSSLTPGFFILNGVPGLEATHIWISLPFCFMYIIAVVGNCGLICLISHEEALHRPMYYFLALLSFTDVTLCTTMVPNMLCIFWFNLKEIDFNACLAQMFFVHMLTGMESGVLMLMALDRYVAICYPLRYATILTNPVIAKAGLATFLRNVMLIIP FTLLTKRLPYCRGNFIPHTYCDHMSVAKVSCGNFKVNAIYGLMVALLIGVFDICCISVSYTMILQAVMSLSSADARHKAFSTCTSHMCSIVITYVAAFFTFFTHRFVGHNIPNHIHIIVANLYLLLPPTTMNPIVYGVKTKQIQEGVIKFLLGDKVSFTYDK OR5T1 DNA-SEQ ID NO:39 atgtttatattaataagcttcacagaagaatttgatgtgcaagtcttcctattttattattttagcaatctatctattcactctaataggcaatttagggctggttgtaccgatcattggggatttctggcttcacagcccaatgtactattttcttggtgttttatcattcttggatgtctgctattctacagttgtcactccaaaaatgttggtcaatttcctg gcaaaaaataaatctatttcatttcttggatgtgcaacacagatgtttcttgcttgtacttttggaaccacagaatgctttctcttggctgcaatggcttatgatcgctatgtagccatctacaaccctctcctgtattcagtgagcatgtcacccagagtctatgtgccactcatcactgcttcctatgttgctagcattttacatgctactatacatacagtggcta catttagcctgtccttctgtggatccaatgaaattaggcatgtcttttgtaatatgcctcctctccttgctatttcttgttctgacactcacgtaatccagctttattcttctactttgtgggctctattgagatagtcactatcctgattgtcctgatctcctatggttttatctgttggccattctgaagatgcagtctgctgaagggaggagaaaagtctttc tacatgtggagctcacctaactggagtgacaatttatcatgggacaatcctcttcatgtatgtgagaccaagttccagctacacttcggacaatgacatgatagtgtcaatattttataccattgtgattcccatgctgaatcccatcatctacagtttgcggaacaaaagatgtaaaggaggcaatcaaaagattgcttgtgagaaattggttcataaataagttatag protein-sequence number40 MFILISFTEEFDVQVFLFLLFLAIYLFTLIGNGLVVPIIGDFWLHSPMYYFLGVLSFLDVCYSTVVTPKMLVNFLAKNKSISFLGCATQMFLACTFGTTECFLLAAMAYDRYVAIYNPLLYSVSMSPRVYVPLITASYVASILHATIHTVA TFSLSFCGSNEIRHVFCNMPPLLAISCSDTHVIQLLFFYFVGSIEIVTILIVLISYGFILLAILKMQSAEGRRKVFSTCGAHLTGVTIYHGTILFMYVRPSSSYTSDNDMIVSIFYTIVIPMLNPIIYSLRNKDVKEAIKRLLVRNWFINKL OR4S2 DNA sequence number 41 atggaaaaaataaacaacgtaactgaattcattttctggggtctttctcagagcccagagattgagaaagtttgttttgtggtgttttctttcttcatataatcattcttctgggaaatctcctcatcatgctgacagtttgcctgagcaacctgtttaagtcacccatgtatttctttctcagcttcttgtcttttgtggacatttgttactcttcagtcacagctcccaag atgattgttgacctgttagcaaaggacaaaaccatctcctatgtggggtgcatgttgcaactgtttggagtacatttctttggttgcactgagatcttcatccttactgtaatggcctatgatcgttatgtggctatctgtaaacccctacattatatgaccatcatgaaccgggagacatgcaataaatgttatagggacgtgggtaggtgggttcttacactccattatc caagtggctctggtagtccaactacccttttgtggacccaatgagatagatcactacttttgtgatgttcaccctgtgttgaaacttgcctgcacagaaacatacattgttggtgttgttgtgacagccaacagtggtaccattgctctggggagttttgttatcttgctaatctcctacagcatcatcctagtttccctgagaaagcagtcagcagaaggcaggcgcaaagcc ctctccacctgtggctcccacattgccatggtcgttatctttttcggcccctgtacttttatgtacatgcgccctgatacgaccttttcagaggataagatggtggcttattttacaccattatcactcccatgttaaatcctctgatttatacactgagaaatgcagaagtaaagaatgcaatgaagaaactgtggggcagaaatgttttcttggaggctaaaggggaaatag protein-sequence number42 MEKINNVTEFIFWGLSQSPEIEKVCFVVFSFFYIIILLGNLLIMLTVCLSNLFKSPMYFFLSFLSFVDICYSSVTAPKMIVDLLAKDKTISYVGCMLQLFGVHFFGCTEIFILTVMAYDRYVAICKPLHYMTIMNRETCNKMLLGTWVGGFLHSI IQVALVVQLPFCGPNEIDHYFCDVHPVLKLACTETYIVGVVVTANSGTIALGSFVILLISYSIILVSLRKQSAEGRRKALSTCGSHIAMVVIFFGPCTFMYMRPDTTFSEDKMVAVFYTIITPMLNPLIYTLRNAEVKNAMKKLWGRNVFLEAKGK FLAG (registered trademark) tag DNA-SEQ ID NO:43 gattacaaggacgacgacgataag Protein - SEQ ID NO: 44 DYKDDDDK Rho Tag DNA-SEQ ID NO:45 atgaacgggaccgagggcccaaacttctacgtgcctttctccaacaagacgggcgtggtg Protein - SEQ ID NO: 46 MNGTEGPNFYVPFSNKTGVV Lucy Tag DNA-SEQ ID NO:47 atgagaccccagatcctgctgctcctggccctgctgacccctaggcctggct Protein - SEQ ID NO: 48 MRPQILLLLALLTLGLA Human G protein alpha subunit G olf DNA-SEQ ID NO:49 Protein - SEQ ID NO:50 MGLCYSLRPLLFGGPGDDPCAASEPPVEDAQPAPAPALAPVRAAARDTARTLLPRGGEGSPACARPKADKPKEKRQRTEQLSAEEREAAKEREAVKEARKVSRGIDRMLRDQKR DLQQTHRLLLLGAGESGKSTIVKQMRILHVNGFNPEEKKQKILDIRKNVKDAIVTIVSAMSTIIPPVPLANPENQFRSDYIKSIAPITDFEYSQEFFDHVKKLWDDEGVKACFER SNEYQLIDCAQYFLERIDSVSLVDYTPTDQDLLRCRVLTSGIFETRFQVDKVNFHMFDVGGQRDERRKWIQCFNDVTAIIYVAACSSYNMVIREDNNTNRLRESLDLFESIWNN RWLRTISIILFLNKQDMLAEKVLAGKSKIEDYFPEYANYTVPEDATPDAGEDPKVTRAKFFIRDLFLRISTATGDGKHYCYPHFTCAVDTENIRRVFNDCRDIIQRMHLKQYELL Human G protein alpha subunit Gα15 DNA-SEQ ID NO:51 Protein - SEQ ID NO:52 MARSLTWRCCPWCLTEDEKAAARVDQEINRILLEQKKQDRGELKLLLLGPGESGKSTFIKQMRIIHGAGYSEEERKGFRPLVYQNIFVSMRAMIEAMERLQIPFSRPESKHHASLVMSQDPYKVTTFEKRYAAAMQWLWRDAGIRACYERRREFHLLDSAVYYLSHLERITEEGYVPTAQDVLRSRM PTTGINEYCFSVQKTNLRIVDVGGQKSERKKWIHCFENVIALIYLASLSEYDQCLEENNQENRMKESLALFGTILELPWFKSTSVILFLNKTDILEEKIPTSHLATYFPSFQGPKQDAEAAKRFILDMYTRMYTGCVDGPEGSKKGARSRRLFSHYTCATDTQNIRKVFKDVRDSVLARYLDEINLL 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 are not intended to limit the scope of the invention described in the Summary, Detailed Description, or Claims. [Brief description of the drawings]

[0089] [Figure 1] FIG. 1 shows the level of pairwise amino acid identity between the receptors referred to herein. [Diagram 2] Figure 2 shows DMTS dose-response curves for mouse odorant receptors Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, and human odorant receptors OR4S2 and OR52N5. [Diagram 3]FIG. 3 shows DMTS dose-response curves for human odorant receptors OR2L13, OR4C15, OR8H3, OR11G2, and OR52N2. [Figure 4] FIG. 4 shows the ranking of volatile compounds that exert specific inhibitory effects on DMTS receptor activity. [Diagram 5] FIG. 5 shows DMTS and DMDS dose-response curves for mouse odorant receptors Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742 and human odorant receptor OR4S2.

[0090] Working Example Example 1 Identification of novel mouse and human DMTS-activated odorant receptors Identification of new odorant receptors was performed according to the method disclosed in WO 2014 / 210585. Briefly, mouse olfactory neurons were exposed to DMTS and Ca 2+Imaging techniques were used to screen. Neurons activated by DMTS were further isolated for complete transcriptome analysis to identify responsive odorant receptors. cDNA corresponding to 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). The amplified cDNA was then used to generate an Illumina cDNA library for next-generation sequencing and 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 could be achieved. Phylogenetic relationship assessment using sequence similarity searches was then used to identify the corresponding human ORs. Similar functional response profiles between orthologous OR pairs have been frequently observed [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 WO 2016 / 201152] and can be used for human OR identification [e.g., Armelin-Correa LM and Malnic B. J Agric Food Chem. doi: 10. 1021 / acs. jafc. 6b04998 (2017)]. Figure 1 shows the pairwise identity levels between the receptors mentioned herein. ORs that do not share orthologous and / or paralogous relationships share an average of only 39% ± 13.1%. All orthologous and paralogous relationships share an average of 77% ± 7.5%. All paralogous and orthologous pairs are shown in grey shaded cells, which represent the highest level of identity supported by the amino acid identity levels shown.

[0091] Example 2 Functional characterization of mouse and human DMTS receptors Functional dose-response experiments were performed to evaluate the level of DMTS activity of the modified cell lines expressing each putative DMTS receptor. Using cell-based assays, mouse receptors Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738 and Olfr742, and human receptors OR4S2 and OR52N5 were tested in HEK293T cell lines, in which the endogenous RTP1 gene was activated and the chaperone proteins of the odorant receptors were expressed (described in WO 2016 / 201153). The mouse receptor genes were tagged with the Lucy-FLAG®-Rho tag combination, and the human receptors were tagged with the Rho-FLAG® tag combination, resulting in tag::receptor fusion proteins in cell-based assays. The receptor genes were tagged with the standard olfactory human G protein alpha subunit G olf The mice were co-transfected with the gene and exposed to increasing concentrations of malodorous odorants. olf Co-expression of Gs activates the Gs transduction pathway, which leads to an increase in internal cAMP upon binding to the appropriate ligand. Odorant-induced activity was detected by measuring cytosolic cAMP increase using a homogeneous time-resolved fluorescence (HTRF)-based kit (CisBio, cAMP dynamic 2 kit, cat.62AM4PEJ). A dose-dependent increase in receptor activity is found specifically for DMTS and 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 for each receptor, as a measure of the EC50 value (effective concentration at which the receptor responds at half the level of the maximal activation effect of a given compound).

[0092] Example 3 Functional characterization of additional human DMTS receptors Functional dose-response experiments were performed to assess the level of DMTS activity of engineered cell lines expressing each putative DMTS receptor. Cell lines stably expressing human receptors were generated in HEK293T cell lines for each of the following human receptors: OR2L13, OR4C15, OR5AC2, OR8H3, OR11G2, or OR52N2. Receptors were tagged with a FLAG®-Rho tag combination and expressed with the human G protein alpha subunit Gα for cell-based assays. 15 It was stably co-expressed with human Gα 15 Co-expression of Ca2+ induces internal Ca2+ expression upon binding to the appropriate ligand. 2+ Receptors were exposed to increasing concentrations of DMTS. A calcium-sensitive fluorescent dye (Molecular Devices, Calcium 5 dye, cat. R8186) was used to measure cytosolic Ca. 2+ The odorant-induced activity was detected by measuring the increase and measuring the change in relative fluorescence ratio (RFU) using a fluorescence imaging plate reader (Molecular Devices, FLIPR) after exposure to odorants. The dose-dependent increase in receptor activity was recorded and the corresponding dose-response curve is shown for DMTS (Figure 2). A cell line lacking receptors was used as a control for non-specific activity at high compound concentrations ("no receptor"). Activity levels are reported by the potency of DMTS-induced response for each receptor as a measure of 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 of the test compounds were presented to the cells. Single-point monitoring of DMTS-induced cellular activity in the presence or absence of test compounds allowed the identification of compounds with putative inhibitory or inhibitory effects. These hits were further confirmed in an inhibition dose-response curve assay that evaluates the potency of activity inhibition as a measure of IC50 (the inhibitor concentration at which receptor activity is inhibited at a level half the maximal inhibitory effect of a given test compound). Dose-dependent reductions in receptor activity were recorded with increasing concentrations of test compound in the presence of a single activating concentration of DMTS (EC80) to obtain corresponding dose-response inhibition curves. The compounds in the table below are examples of compounds that reduced DMTS-induced activity of at least one receptor, as shown in Figure 4. [Table 2]

[0094] In FIG. 4, the length of the bars indicates the potency (IC50, expressed as negative log molar concentration) of the activity inhibition of the selected compound for each receptor. The absence of a bar indicates the absence of inhibition of the corresponding receptor-compound pair. These compounds can specifically bind and block the activity of DMTS or DMDS receptors, and thus suppress or inhibit the odor of DMTS or DMDS. Therefore, such compounds can be used as malodor neutralizers for toilet and breath odor control applications.

[0095] Example 5 DMDS-induced response profiles of mouse and human DMTS receptors Functional dose-response experiments were performed to confirm DMTS activity on the DMTS receptors identified in Example 2 and to further characterize their response to DMDS. Using the same cell-based assay described in Example 2, mouse receptors Olfr1193, Olfr1093, Olfr1097, Olfr166, Olfr169, Olfr738, Olfr742, and human odorant receptor OR4S2 were tested with increasing concentrations of DMTS, DMDS, or butyric acid (control). A strong dose-dependent increase in receptor activity is observed for DMTS and DMDS relative to the butyric acid control compound. A weak response of OR4S2 and Olfr169 to butyric acid was seen, but was too weak to consider butyric acid as a representative ligand for these receptors. Activity levels were reported as EC50 values, EC50 DMTS and EC50 DMDS The potency of the DMTS- or DMDS-induced response of each receptor, respectively, is reported as a measure of unpleasantness. Mock-transfection control experiments, in which cells do not express the odorant receptor, showed no activity upon exposure to DMTS or DMDS. Competitive antagonists at the odor-binding sites of these receptors could reduce the unpleasant perception of both DMTS and DMDS.

Claims

1. 1. A method for identifying a compound that suppresses, blocks, inhibits, and / or modulates the activity of an olfactory receptor activated by a substance causing a malodor, comprising: a. contacting the test substance and the substance causing the malodour with the olfactory receptor, Olfr1097; b. measuring the response of the olfactory receptors to the malodor-causing substance by measuring the response of the olfactory receptors 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 said identified test substance as a compound that modulates the response of said olfactory receptor to said malodor-causing substance. Including, wherein the malodor-causing substance is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

2. 1. A method for identifying a malodor control agent, comprising: a. contacting a test substance and a substance causing a malodor with at least one olfactory receptor, said receptor comprising a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; b. measuring the response of said olfactory receptor polypeptide to said malodor-causing substance; c. identifying a test substance capable of inhibiting 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 suppressant. Including, wherein the substance causing the malodor is DMTS or DMDS.

3. 1. A method for identifying a compound that suppresses, blocks, inhibits, and / or modulates the activity of at least one olfactory receptor activated by a substance causing a malodor, comprising: a. contacting the receptor, or chimera or fragment thereof, with a compound; and b. determining whether said compound affects the activity of said receptor Including, wherein the receptor is i) is a polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; or ii) a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5, or its reverse complement; The method, wherein the substance causing the malodor is dimethyl trisulfide (DMTS) or dimethyl disulfide (DMDS).

4. 1. A method for identifying a compound capable of suppressing or inhibiting the odor of DMTS or DMDS, comprising: (i) contacting a cell line expressing a DMTS or DMDS receptor polypeptide with at least one compound; (ii) screening for compounds that suppress, block, inhibit, and / or modulate the activity of said DMTS or DMDS receptor polypeptide; and (iii) identifying a compound that, when it suppresses, blocks, inhibits, and / or modulates the activity of said DMTS or DMDS receptor polypeptide, is capable of suppressing or inhibiting the odor of DMTS or DMDS, wherein the receptor polypeptide is a. comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6; or b. The method is encoded by a nucleic acid molecule comprising a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5 or its reverse complement.

5. Use of a polypeptide that can be activated by DMTS or DMDS, comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:6, to identify malodor-controlling compounds.

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