Method for assigning olfactory notes to olfactory receptor activation and method for identifying compounds having the assigned notes

Abstract

The present invention relates to the fragrance industry. More specifically, the present invention relates to assays and methods for screening and identifying compositions and / or ingredients that enhance a subject's perception of a target odorant compound based on the use of specific olfactory receptors activated by the target odorant compound.

Description

[Technical Field]

[0001] The present invention relates to the fields of olfactory receptors and aroma receptors and odorant assays that can be used to identify compounds having at least one odor note.

[0002] Background of the Invention Since the first identification of odorant receptors in 1991, much has been done to improve our understanding of olfaction and how humans perceive volatile chemicals. At the periphery of the human olfactory system, the main olfactory epithelium lining the nasal cavity houses millions of olfactory sensory neurons (OSNs), each expressing a single member from a family of approximately 400 intact odorant receptor (OR) genes. Each OR can be activated by several volatile compounds, or conversely, a single volatile compound can activate several ORs. This results in a combinatorial OSN activation code for each volatile chemical and its mixtures. Each receptor is stochastically expressed in OSNs in a monogenic and monoallelic manner, resulting in one OSN type for each OR allele present in the genome. Depending on the frequency of genetic selection for each OR, distinct subsets of the OSN population specialize in each OSN type. Each of these OSN type subpopulations provides a distinct channel of information about the molecular identity, or identity, and concentration of the volatile compound with which it is in contact at any given time. The level of activity induced in each OSN of each OSN type varies according to the concentration of volatile compounds to which that OR is receptive, as well as according to the binding and activation parameters associated with each OR-compound pair. Furthermore, some OR-ligand pairs induce or enhance OSN activity, whereas others reduce or prevent it. This can occur competitively, when compounds bind to the OR, or noncompetitively, when multiple compounds can simultaneously bind to the OR. The resulting combinatorial logic of these approximately 400 input channels (i.e., the peripheral olfactory system) is transduced by subsequent downstream neural networks, resulting in the sense of smell.

[0003] The Fragrance & Flavor (F&F) industry is constantly searching for new ingredients, novel fragrance and flavor applications, improved sensory experiences, and more stable, biodegradable, and non-toxic compounds.

[0004] Because an odorant or aroma molecule may interact with several olfactory receptors (ORs), it is often difficult to infer the odor notes of a volatile compound based solely on its chemical structure. Conversely, because each OR may interact with several odorants or aroma molecules with different chemical structures, eliciting a different set of odor notes, it is often difficult to infer the nature of the information encoded by the ORs.

[0005] There remains a need to predict at least one olfactory note of a given compound or composition based on OR activation, and to facilitate the discovery of new fragrance and flavor ingredients relevant to the F&F industry.

[0006] Summary of the Invention In one aspect, a method for associating at least one olfactory note with an olfactory receptor is provided, the method comprising: (a) providing an olfactory receptor; (b) contacting the olfactory receptor with a compound having at least one known odor note; (c) determining whether the compound activates an olfactory receptor; (d) repeating steps (b) and (c) with at least one compound having a known odor note, wherein the compound in step (d) is different from the compound in steps (b) and (c) of the preceding iteration; (e) classifying the compounds from steps (b)-(d) that activate olfactory receptors into a subset; (f) identifying at least one olfactory note common to the subset of compounds; (g) assigning at least one identified odor note to an olfactory receptor; Includes.

[0007] In one aspect, there is provided a method for screening at least one compound having a particular odor note, the method comprising: (a) providing an olfactory receptor having at least one identified odor note; (b) contacting the olfactory receptor with at least one compound; (c) determining whether the at least one compound activates an olfactory receptor; (d) associating the at least one compound with the at least one identified odor note if the at least one compound activates an olfactory receptor; Includes.

[0008] In some embodiments, a plurality of olfactory receptors, each having at least one different identified odor note, is provided in step (a), and the plurality of identified odor notes are associated in step (d) with a compound or combination of compounds that activates the plurality of olfactory receptors according to steps (b) and (c) in the above embodiments.

[0009] In one aspect, a method for screening at least one compound for an arthy tone is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating an AR activity with the at least one compound if the at least one compound activates the polypeptide; and wherein the polypeptide is an olfactory receptor.

[0010] In one aspect, a method for screening at least one compound for coumarin activity is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a coumarin moiety with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0011] In one aspect, a method for screening at least one compound for lactonic coconut flavor is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating the at least one compound with a lactonic coconut flavor if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0012] In one aspect, a method for screening at least one compound for fenugreek flavor is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a fenugreek extract with the at least one compound if the at least one compound activates the polypeptide; and wherein the polypeptide is an olfactory receptor.

[0013] In one aspect, a method is provided for screening at least one compound for a powdery musk note, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a powdery musk note with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0014] In one aspect, a method is provided for screening at least one compound for an animal musk note, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating an animalic musk note with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0015] In one aspect, a method is provided for screening at least one compound for violet hue, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a violet hue with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0016] In one aspect, a method for screening at least one compound for blonde wood tone is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a blond wood odor note with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0017] In one aspect, a method for screening at least one compound for lily of the valley tone is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating the at least one compound with a lily of the valley tone if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0018] In one aspect, a method for screening at least one compound for linalic activity is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29; (b) contacting the polypeptide with at least one compound; (c) determining whether the compound or combination of compounds activates the polypeptide; (d) associating a linalic acid with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0019] In one aspect, a method for screening at least one compound for a jasmine note is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31; (b) contacting the polypeptide with at least one compound; (c) determining whether the compound or combination of compounds activates the polypeptide; (d) associating a jasmine note with the at least one compound if the at least one compound activates the polypeptide; and wherein the polypeptide is an olfactory receptor.

[0020] In some embodiments, the present disclosure provides at least one compound identified by a method according to certain embodiments described herein.

[0021] In one aspect, a method for replacing a compound in a scented composition is provided, the method comprising: (a) selecting a compound in a composition having a scent; (b) identifying the odor notes of the compounds in the composition; (c) providing olfactory receptors associated with the olfactory notes; (d) contacting the olfactory receptor with a test compound; (e) determining whether the test compound activates an olfactory receptor; (f) if the test compound activates the olfactory receptor, replacing the selected compound in the composition with the test compound; Includes.

[0022] In one aspect, a method is provided for replacing at least one compound in a plurality of compounds in a scented composition, the method comprising: (a) selecting a plurality of compounds in a scented composition, wherein each compound in the plurality of compounds has at least one scent note; (b) identifying the odor notes of a plurality of compounds; (c) providing a plurality of olfactory receptors, wherein two or more of the olfactory receptors are assigned the same odor note as at least one compound of the plurality of compounds; (d) contacting at least one test compound with two or more of the olfactory receptors; (e) determining whether at least one test compound activates two or more olfactory receptors; (f) if the test compound activates the same olfactory receptors as two or more compounds of the plurality of compounds, replacing two or more compounds of the plurality of compounds with the test compound; Includes.

[0023] In one aspect, a method for producing a composition having a desired fragrance is provided, the method comprising: (a) selecting a desired scent; (b) determining a plurality of notes that combine to produce a desired scent; (c) adding at least one compound having a plurality of fragrance notes to the composition; Includes.

[0024] In some embodiments, at least one compound having multiple odor notes is identified by a method according to certain embodiments described herein.

[0025] In one aspect, a method for removing at least one compound in a composition having a desired scent is provided, the method comprising: (a) selecting a composition having a desired scent, wherein the scent is comprised of at least one fragrance note; (b) identifying at least one olfactory receptor associated with at least one odor note; (c) selecting at least one compound in the composition that has a desired scent; (d) contacting at least one compound with at least one olfactory receptor; (e) determining whether the compound inhibits at least one olfactory receptor; (f) removing the at least one compound from the composition having the desired scent if the at least one compound inhibits the at least one olfactory receptor; Includes.

[0026] In one aspect, a method for adding at least one compound to a scented composition is provided, the method comprising: (a) selecting a composition having a fragrance, wherein the fragrance is comprised of at least one fragrance note; (b) identifying at least one olfactory receptor associated with at least one odor note; (c) contacting at least one olfactory receptor with at least one test compound; (d) determining whether the at least one test compound inhibits at least one olfactory receptor; (e) adding at least one test compound to a composition having a fragrance if the at least one test compound inhibits at least one olfactory receptor; wherein at least one olfactory receptor is assigned an undesirable odor note in the fragrance.

[0027] In one aspect, a method for adding at least one compound to a scented composition is provided, the method comprising: (a) selecting a composition having a fragrance, wherein the fragrance is comprised of at least one fragrance note; (b) identifying a first olfactory receptor associated with a first scent note of the at least one scent note, wherein the first scent note is desired in a fragrance; (c) contacting the first olfactory receptor with a test compound; (d) determining whether the test compound activates the first olfactory receptor; (e) identifying a second olfactory receptor associated with a second of the at least one scent note, wherein the second scent note is undesired in the fragrance; (f) contacting the second olfactory receptor with a test compound; (g) determining whether the test compound inhibits a second olfactory receptor; (h) if the test compound activates the first olfactory receptor and inhibits the second olfactory receptor, adding the test compound to a composition having a scent; Includes.

[0028] In some embodiments, the added test compound replaces a compound in the fragrance. [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 summarizes how a particular volatile compound acquires at least one olfactory note via OR activation. Compounds that activate multiple receptors exhibit corresponding notes that are associated with the activated ORs. [Figure 2]FIG. 1 shows the correlation between OR activation and odor notes, particularly around receptors OR5AU1 (associated with coconut notes), OR8B3 (associated with coumarin notes), and OR8D1 (associated with fenugreek notes). [Figure 3] Figure 1 shows the human odorant receptor OR11A1 activation ranking obtained from a single-concentration high-throughput screening process using a large and diverse set of volatile compounds. [Figure 4] This figure shows the results of dose-response experiments on the human odorant receptor OR11A1 obtained for four active agonists (geosmin, vulcanolide, fenchyl alcohol, and patchouli alcohol), capturing both the potency and efficacy of each compound on the receptor. [Figure 5] FIG. 4 shows the molecular acceptance range OR11A1 according to potency and efficacy obtained from dose-response experiments with hit compounds from FIG. 3. The most active agonists are highlighted. [Figure 6] FIG. 1 shows the chemical structures of the four most active agonists and the corresponding agonists of partial agonists of OR11A1. [Figure 7] FIG. 1 shows the molecular spectrum of OR8B3 agonists according to potency and efficacy, highlighting the most active agonists that share a coumarin moiety. [Figure 8] FIG. 1 shows the molecular spectrum of OR5AU1 agonists according to potency and efficacy, highlighting the most active agonists with a common lactonic coconut flavor. [Figure 9] FIG. 1 shows the molecular spectrum of OR8D1 agonists according to potency and efficacy, highlighting the most active agonists with a common fenugreek flavor. [Figure 10] FIG. 1 shows the molecular envelope of OR5AN1 agonists according to potency and efficacy, highlighting the most active agonists that share a powdery musk note. [Figure 11]FIG. 1 shows the molecular spectrum of OR1N2 agonists according to potency and efficacy, highlighting the most active agonists that share an animalic musk note. [Figure 12] FIG. 1 shows the molecular spectrum of OR5A1 agonists according to potency and efficacy, highlighting the most active agonists with a common violet hue. [Figure 13] FIG. 1 shows the molecular envelope of OR7A17 agonists according to potency and efficacy, highlighting the most active agonists that share a blond wood tone. [Figure 14] FIG. 1 shows the molecular acceptance spectrum of OR10J5 agonists according to potency and efficacy, highlighting the most active agonists with a common lily of the valley tone. [Figure 15] FIG. 1 shows the molecular spectrum of OR1C1 agonists according to potency and efficacy, highlighting the most active agonists with a common linalinic profile. [Figure 16] FIG. 1 shows the molecular spectrum of OR5B12 agonists according to potency and efficacy, highlighting the most active agonists that share a jasmine note. [Figure 17] FIG. 1 shows the activity levels of components that activate OR8D1, OR8B3, and OR5AU1 in percentage. [Figure 18A] Figure 1 shows the odorant receptor activity induced by validation of Accords A and B by comparing the model with in vitro control experiments. [Figure 18B] Figure 1 shows the odorant receptor activity induced by validation of Accords A and B by comparing the model with in vitro control experiments. [Figure 18C] Figure 1 shows the odorant receptor activity induced by validation of Accords A and B by comparing the model with in vitro control experiments. [Figure 19] FIG. 10 shows the agreement between flavor evaluations for celery notes and sensory predictions from the model. [Figure 20A]Figure 1 shows the odorant receptor activity induced by validation of Accords A, C and D by comparing the model with in vitro control experiments. [Figure 20B] Figure 1 shows the odorant receptor activity induced by validation of Accords A, C and D by comparing the model with in vitro control experiments. [Figure 20C] Figure 1 shows the odorant receptor activity induced by validation of Accords A, C and D by comparing the model with in vitro control experiments. [Figure 21] FIG. 1 shows the agreement between perfume ratings on Hay notes and sensory predictions from the model. [Figure 22] FIG. 1 shows the agreement between fragrance ratings and sensory predictions from the model for coconut notes.

[0030] Detailed Description definition As used herein, the term "OR" or "olfactory receptor" or "olfactory substance receptor" refers to one or more members of a family of G protein-coupled receptors (GPCRs) expressed in olfactory cells. Also, OSNs (olfactory receptor cells) can be identified based on morphology or by the expression of proteins specifically expressed in olfactory cells. Members of the OR family may have the ability to act as receptors for olfactory signal transduction.

[0031] An "agonist of an OR" or "agonist compound" refers to a volatile compound or ligand that binds to an OR, activates the OR, and induces the olfactory receptor transduction cascade.

[0032] "Efficacy" refers to the measure of receptor activity induced by agonist (odorant) binding, in terms of the amount or concentration required to produce a given level of activity. This indicates the sensitivity of the receptor to different agonist concentrations and is often referred to as the EC 50 The agonist activity is evaluated by calculating the agonist concentration required to achieve half-maximal receptor activity.

[0033] "Efficacy" refers to a measure of the level of activity an OR responds to a given agonist, and is obtained by measuring the activation span between constitutive activity (baseline in the absence of agonist) and agonist-induced activity.

[0034] The "receptive field" or "molecular receptive range" of an odorant receptor refers to the range of volatile compounds that activate the receptor. This refers to the set of different volatile compounds that can bind to the receptor, trigger a transduction cascade within the cell, and transmit the chemical stimulus via the olfactory neuron to the brain.

[0035] OR or odorant receptor or olfactory receptor polypeptides belong to the seven-transmembrane domain G protein-coupled receptor superfamily, encoded by a single exon approximately 1 kb long, and are considered as such if they exhibit characteristic olfactory receptor-specific amino acid motifs. The seven domains are termed "transmembrane" or "TM" domains TM I-TM VII, connected by three "intracellular loop" or "IC" domains IC I-IC III, and three "extracellular loop" or "EC" domains EC I-EC III. The motifs and their variants are defined as, but not limited to, the presence of the MAYDRYVAIC motif (SEQ ID NO: 7) that overlaps with TM III and IC II, the FSTCSSH motif (SEQ ID NO: 8) that overlaps with IC III and TM VI, the PMLNPFIY motif (SEQ ID NO: 9) in TM VII as well as three conserved C residues in EC II, and the highly conserved GN residue in TM I [Zhang, X. & Firestein, S. Nat. Neurosci. 5, 124-133 (2002); Malnic, B., et al. Proc. Natl. Acad. Sci. USA 101, 2584-2589 (2004)].

[0036] Class I and Class II ORs refer to phylogenetically distinct classes of odorant receptor GPCRs. Class I ORs are more closely related to ORs predominant in aquatic species. Class II ORs are more closely related to terrestrial species. Mammals possess both types.

[0037] As used herein, "olfactory note" or "olfactory note" refers to a specific olfactory perception and refers to the activation of ORs by a compound. Non-limiting examples of olfactory notes include citrus, coconut, patchouli, etc. A compound may have two or more odor notes. For example, as shown in Figure 1, compound (1) (β-ionone) has violet and blond woody notes resulting from the activation of OR5A1 and OR7A17.

[0038] As used herein, "aroma" refers to the olfactory perception resulting from the sum of the activation, enhancement, and inhibition of olfactory receptors (if present) by at least one compound. Thus, by way of example and not intended to limit the scope of the present disclosure in any way, an "aroma" may result from the olfactory perception resulting from the sum of a first compound that activates ORs associated with coconut notes, a second compound that activates ORs associated with celery notes, and a third compound that inhibits ORs associated with camphor notes.

[0039] As used herein, "note" or "olfactory note" or "fragrance note" identifies an olfactory category. For example, as shown in Figure 1, floral notes include lily of the valley and violet notes.

[0040] OR nucleic acids encode the GPCR family, which has seven transmembrane domains and has G protein-coupled receptor activity, e.g., it couples to G proteins in response to extracellular stimuli and catalyzes the production of IP3, cAMP, cGMP, and Ca via the stimulation of enzymes such as phospholipase C and adenylate cyclase III. 2+ It may promote the production of intracellular second messengers such as

[0041] The "N-terminal domain" region begins at the N-terminus (amino terminus) of a peptide or protein and extends to the region near the start of the first transmembrane domain.

[0042] The "transmembrane region" includes the seven "transmembrane domains," which refer to the domains of the OR polypeptide that are located 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 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. Therefore, the primary structural sequence can be predicted based on the known transmembrane domain sequence. These transmembrane domains are useful for in vitro ligand binding assays.

[0043] The term "functional effect" in the context of assays for testing compounds that modulate olfactory transduction via OR family members includes the measurement of any parameter that is indirectly or directly under the influence of the receptor, e.g., functional, physical, and chemical effects, including ligand binding, 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). 2+ ), including in vitro, in vivo, and ex vivo changes, as well as other physiological effects such as increased or decreased neurotransmitter or hormone release.

[0044] "Measuring functional effects" or "confirming activity" in the context of an assay refers to assaying a compound to indirectly or directly increase or decrease parameters, such as functional, physical, and chemical effects, that are influenced by an OR family member. Such functional effects can be measured by any means known to those skilled in the art, such as changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamping, membrane voltage-sensitive dyes, whole-cell currents, radioisotope flux, inducible markers, oocyst 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; neurotransmitter release, etc.

[0045] As used herein, the terms "purified," "substantially purified," and "isolated" refer to a state in which the compounds of the invention are free from other heterologous compounds with which they are normally associated in their natural state; thus, "purified," "substantially purified," and "isolated" refer to compounds of the invention that comprise at least 0.5%, 1%, 5%, 10%, or 20% by weight of the mass of a given sample, or at least 50% or 75% by weight of the mass of a given sample. In a specific embodiment, these terms refer to compounds of the invention that comprise at least 95%, 96%, 97%, 98%, 99%, or 100% by weight of the mass of a given sample. As used herein, the terms "purified," "substantially purified," and "isolated" of a nucleic acid or protein, when referring to a nucleic acid or protein, also refer to a state of purification or concentration that differs from that which occurs naturally in a mammal, particularly a human. Any degree of purification or concentration greater than that which occurs naturally in the mammalian, especially human, body is within the meaning of "isolated," including (1) purification from other related structures or compounds, or (2) association with structures or compounds not normally associated in the mammalian, especially human, body. The nucleic acids or proteins or classes of nucleic acids or proteins described herein may be isolated or otherwise associated with structures or compounds not normally associated in nature according to a variety of methods and processes known to those of skill in the art.

[0046] The term "nucleic acid" or "nucleic acid sequence" refers to deoxyribonucleotide or ribonucleotide oligonucleotides in either single- or double-stranded form. The term encompasses nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved, for example, by generating sequences in which the third position of one or more selected codons is substituted with mixed-base and / or deoxyinosine residues.

[0047] In addition to the genetic sequences shown in the sequences disclosed herein, variants also include DNA sequence polymorphisms that may exist within a given population, which may result in changes to the amino acid sequences of the polypeptides disclosed herein. Such genetic polymorphisms may exist within a population due to cells from different populations or natural allelic variations. Allelic variants may also include functional equivalents.

[0048] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, whether or not it contains naturally occurring and non-naturally occurring amino acids or polymers. The term "heterologous," when used with 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, having 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).

[0049] "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, e.g., cultured cells, explants, and in vitro cells.

[0050] A "tag" or "tag combination" refers to a short polypeptide sequence that can be added to an odorant receptor protein. Typically, DNA encoding the "tag" or "tag combination" is added to DNA encoding the receptor, 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 transport to the cell membrane, thus supporting 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)].

[0051] 2-16, the present invention includes methods for identifying olfactory perception commonalities between chemically and organoleptically diverse volatile compounds based on OR activation profiles. While not intending to be limited to a particular theory, OR activation profiles represent a better predictor of the olfactory notes of a given volatile compound compared to physicochemical similarity alone. While not intending to be limited to a particular theory, physicochemical similarity may only partially predict olfactory similarity.

[0052] The present invention includes methods by which at least one olfactory note can be associated with an OR by screening a library of chemically and organoleptically diverse volatile compounds against the OR and identifying at least one olfactory note common to the screened volatile compounds that are activators of the OR.

[0053] Thus, in one aspect, the present invention provides a method for providing an OR, contacting the OR with a volatile compound having at least one known odor note, determining whether the compound activates at least one olfactory receptor, repeating the contacting and determining steps with a different compound having at least one known odor note, classifying a subset of compounds that activate the OR, identifying at least one known odor note that is common to the subset of compounds, and associating the identified at least one known odor note with the OR.

[0054] In another aspect, a method is provided that includes contacting an OR associated with the identified at least one known odor note with at least one volatile compound, determining whether the at least one compound activates the OR, and if the at least one volatile compound activates the OR, associating the identified at least one known odor note associated with the OR with the at least one volatile compound.

[0055] In some embodiments of the present disclosure, the method is used to decode an olfactory code for a particular olfactory note by assessing olfactory receptor activity induced by at least one volatile compound and associating the resulting olfactory note based on the observed olfactory receptor activity.

[0056] The olfactory notes of a volatile compound having two or more olfactory notes can be predicted from the activation of multiple olfactory receptors by the volatile compound.Furthermore, according to the present invention, the olfactory notes of a mixture of volatile compounds can be predicted from the activation of multiple olfactory receptors by the mixture.In some embodiments, the mixture comprises multiple volatile compounds.

[0057] In one aspect, the volatile compounds exhibit several olfactory notes that can be predicted by assessing the olfactory receptor activity induced by the compound and correlating the resulting olfactory notes based on their activity on multiple ORs.

[0058] In another embodiment, the olfactory notes linked to the activity of an OR can be correlated by probing the receptor's receptivity, i.e., generating functional activation data to create a comprehensive list of its activators, and then identifying the most common olfactory notes among them.

[0059] In a further aspect, the common olfactory notes of compounds that activate a given receptor can be related by comparing the overall descriptions of the compounds and identifying common descriptors between activators.It should be understood that such olfactory notes can be semantically described in several ways, for example, by perfumers.Examples of such semantic similarities that capture similar olfactory notes include, for example, marine, watery and ozone notes; earthy, humus and moss notes; hay, coumarin and tonka; celery, fenugreek and maple; lily of the valley and lily of the valley.

[0060] In one aspect, a method for associating at least one olfactory note with an olfactory receptor is provided, the method comprising: (a) providing an olfactory receptor; (b) contacting the olfactory receptor with a compound having at least one known odor note; (c) determining whether the compound activates an olfactory receptor; (d) repeating steps (b) and (c) with at least one compound having a known odor note, wherein the compound in step (d) is different from the compound in steps (b) and (c) of the preceding iteration; (e) classifying the compounds from steps (b)-(d) that activate olfactory receptors into a subset; (f) identifying at least one olfactory note common to the subset of compounds; (g) assigning at least one identified odor note to an olfactory receptor; Includes.

[0061] In one aspect, there is provided a method for screening at least one compound having a particular odor note, the method comprising: (a) providing an olfactory receptor having at least one identified odor note; (b) contacting the olfactory receptor with at least one compound; (c) determining whether the at least one compound activates an olfactory receptor; (d) associating the at least one compound with the at least one identified odor note if the at least one compound activates an olfactory receptor; Includes.

[0062] In some embodiments, a plurality of olfactory receptors, each having at least one different identified odor note, is provided in step (a), and the plurality of identified odor notes are associated in step (d) with a compound or combination of compounds that activates the plurality of olfactory receptors according to steps (b) and (c) in the above embodiments.

[0063] In one aspect, a method for screening at least one compound for an arthy tone is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating an AR activity with the at least one compound if the at least one compound activates the polypeptide; and wherein the polypeptide is an olfactory receptor.

[0064] In one aspect, a method for screening at least one compound for coumarin activity is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a coumarin moiety with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0065] In one aspect, a method for screening at least one compound for lactonic coconut flavor is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating the at least one compound with a lactonic coconut flavor if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0066] In one aspect, a method for screening at least one compound for fenugreek flavor is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a fenugreek extract with the at least one compound if the at least one compound activates the polypeptide; and wherein the polypeptide is an olfactory receptor.

[0067] In one aspect, a method for screening at least one compound for a powdery musk note is provided, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a powdery musk note with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0068] In one aspect, a method is provided for screening at least one compound for an animal musk note, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating an animalic musk note with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0069] In one aspect, a method is provided for screening at least one compound for violet hue, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a violet hue with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0070] In one aspect, a method for screening at least one compound for blonde wood tone is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating a blond wood odor note with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0071] In one aspect, a method for screening at least one compound for lily of the valley tone is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27; (b) contacting the polypeptide with at least one compound; (c) determining whether the at least one compound activates the polypeptide; (d) associating the at least one compound with a lily of the valley tone if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0072] In one aspect, a method for screening at least one compound for linalic activity is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29; (b) contacting the polypeptide with at least one compound; (c) determining whether the compound or combination of compounds activates the polypeptide; (d) associating a linalic acid with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

[0073] In one aspect, a method for screening at least one compound for a jasmine note is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31; (b) contacting the polypeptide with at least one compound; (c) determining whether the compound or combination of compounds activates the polypeptide; (d) associating a jasmine note with the at least one compound if the at least one compound activates the polypeptide; and wherein the polypeptide is an olfactory receptor.

[0074] In some embodiments, the present disclosure provides at least one compound identified by a method according to certain embodiments described herein.

[0075] In another aspect, a method is provided for replacing volatile compounds in a scented composition by selecting compounds in the scented composition, identifying at least one known scent note of the compounds in the composition, providing an OR associated with the at least one known scent note, contacting the OR with a test compound, determining whether the test compound activates an olfactory receptor, and if the test compound activates the OR, replacing compounds of the identified at least one scent note in the composition with the test compound.

[0076] In another embodiment, a method is provided for replacing two or more compounds in a scented composition with a single test compound by identifying known scent notes of two or more compounds in the composition to be replaced, providing ORs associated with the known scent notes, contacting the ORs with a single test compound, and if the test compound activates the same ORs as the two or more compounds in the fragrance, replacing the two or more compounds in the composition with the test compound. As a hypothetical example, a scented composition includes compound A having a coconut note, compound B having a celery note, and compound C having a citrus note. Test compound X activates ORs associated with coconut and celery notes. Test compound X replaces compounds A and B in the scented composition.

[0077] In another aspect, a method is provided for creating a composition having a desired scent by selecting a desired scent, determining a plurality of scent notes that combine to create the desired scent, and combining one or more compounds having the plurality of scent notes in the composition.

[0078] In a further aspect of the present invention, there is provided a method for removing a compound having at least one known odor note in a composition having a desired scent, the method comprising: selecting a composition having a desired scent (wherein the at least one known odor note is undesirable or imparts an undesirable odor note to the scent); identifying at least one olfactory receptor associated with the at least one known undesirable odor note; selecting a compound in the composition having the desired scent; contacting the compound with the olfactory receptor; determining whether the compound inhibits the olfactory receptor; and, if the compound inhibits the olfactory receptor, removing the compound from the composition.

[0079] In a further aspect, a method for adding a compound to a scented composition is provided, the method comprising: selecting a scented composition (wherein the added compound inhibits at least one olfactory receptor associated with at least one known undesirable odor note, the scent being composed of at least one known odor note); identifying at least one olfactory receptor associated with the at least one undesirable odor note; contacting the at least one olfactory receptor with a test compound; determining whether the test compound inhibits the olfactory receptor; and, if the test compound inhibits the olfactory receptor, adding the test compound to the scented composition.

[0080] According to the present invention, a method for adding or replacing a compound to a scented composition is provided by the following steps: selecting a scented composition, wherein the scent is composed of at least one scent note; identifying a first olfactory receptor associated with a first of the at least one scent note, wherein the first scent note is desired in the fragrance; contacting the first olfactory receptor with a test compound; determining whether the test compound activates the first olfactory receptor; identifying a second olfactory receptor associated with a second of the at least one scent note, wherein the second scent note is undesired in the fragrance; contacting the second olfactory receptor with the test compound; determining whether the test compound inhibits the second olfactory receptor; and adding the test compound to the scented composition if the test compound activates the first olfactory receptor and inhibits the second olfactory receptor.

[0081] In one aspect, a method for replacing a compound in a scented composition is provided, the method comprising: (a) selecting a compound in a composition having a scent; (b) identifying the odor notes of the compounds in the composition; (c) providing olfactory receptors associated with the olfactory notes; (d) contacting the olfactory receptor with a test compound; (e) determining whether the test compound activates an olfactory receptor; (f) if the test compound activates the olfactory receptor, replacing the selected compound in the composition with the test compound; Includes.

[0082] In one aspect, a method is provided for replacing at least one compound in a plurality of compounds in a scented composition, the method comprising: (a) selecting a plurality of compounds in a scented composition, wherein each compound in the plurality of compounds has at least one scent note; (b) identifying the odor notes of a plurality of compounds; (c) providing a plurality of olfactory receptors, wherein two or more of the olfactory receptors are assigned the same odor note as at least one compound of the plurality of compounds; (d) contacting at least one test compound with two or more of the olfactory receptors; (e) determining whether at least one test compound activates two or more olfactory receptors; (f) if the test compound activates the same olfactory receptors as two or more compounds of the plurality of compounds, replacing two or more compounds of the plurality of compounds with the test compound; Includes.

[0083] In one aspect, a method for producing a composition having a desired fragrance is provided, the method comprising: (a) selecting a desired scent; (b) determining a plurality of notes that combine to produce a desired scent; (c) adding at least one compound having a plurality of fragrance notes to the composition; Includes.

[0084] In some embodiments, at least one compound having multiple odor notes is identified by a method according to certain embodiments described herein.

[0085] In one aspect, a method for removing at least one compound in a composition having a desired scent is provided, the method comprising: (a) selecting a composition having a desired scent, wherein the scent is comprised of at least one fragrance note; (b) identifying at least one olfactory receptor associated with at least one odor note; (c) selecting at least one compound in the composition that has a desired scent; (d) contacting at least one compound with at least one olfactory receptor; (e) determining whether the compound inhibits at least one olfactory receptor; (f) removing the at least one compound from the composition having the desired scent if the at least one compound inhibits the at least one olfactory receptor; Includes.

[0086] In one aspect, a method for adding at least one compound to a scented composition is provided, the method comprising: (a) selecting a composition having a fragrance, wherein the fragrance is comprised of at least one fragrance note; (b) identifying at least one olfactory receptor associated with at least one odor note; (c) contacting at least one olfactory receptor with at least one test compound; (d) determining whether the at least one test compound inhibits at least one olfactory receptor; (e) adding at least one test compound to a composition having a fragrance if the at least one test compound inhibits at least one olfactory receptor; wherein at least one olfactory receptor is assigned an undesirable odor note in the fragrance.

[0087] In one aspect, a method for adding at least one compound to a scented composition is provided, the method comprising: (a) selecting a composition having a fragrance, wherein the fragrance is comprised of at least one fragrance note; (b) identifying a first olfactory receptor associated with a first scent note of the at least one scent note, wherein the first scent note is desired in a fragrance; (c) contacting the first olfactory receptor with a test compound; (d) determining whether the test compound activates the first olfactory receptor; (e) identifying a second olfactory receptor associated with a second of the at least one scent note, wherein the second scent note is undesired in the fragrance; (f) contacting the second olfactory receptor with a test compound; (g) determining whether the test compound inhibits a second olfactory receptor; (h) if the test compound activates the first olfactory receptor and inhibits the second olfactory receptor, adding the test compound to a composition having a scent; Includes.

[0088] In some embodiments, the added test compound replaces a compound in the fragrance.

[0089] The present disclosure further includes compounds identified by the methods described herein.

[0090] Provided by the present disclosure are methods for identifying compounds with desired olfactory notes that characterize relevant receptors and can be used in fragrance or flavor applications to achieve the expected sensory outcome.

[0091] For each OR in the human OR repertoire, several alleles may exist. Each allele contains different single nucleotide polymorphisms (SNPs) that may or may not affect the receptive range of a given OR. Thus, OR allelic variation can add or remove compounds, altering the absolute and relative potency and efficacy of compounds in the list of activated compounds, and thus generating different flavor links for individual OR alleles. Similarly, other allelic differences, including, for example, copy number variation (CNV) or coding sequence truncation, can also result in changes in OR flavor specificity.

[0092] Semantic variation can be addressed, for example, by collecting, curating, and analyzing multiple sources of sensory and chemical descriptions of compounds and chemical mixtures, as well as psychophysical assessments to determine characteristics of given compounds and mixtures, such as quality, intensity, and associated emotions. Implementation of artificial intelligence algorithms or techniques, including but not limited to natural language processing, graph convolutional networks, deep neural networks, and other machine learning approaches, as well as statistical analysis of descriptor similarity and / or co-occurrence or potential mutual exclusivity, can also be applied to mitigate the confounding effects of semantic differentiation. Automated processes can be implemented to associate ORs with aroma notes using probability estimates and correlation scores of descriptor occurrence and measures of receptor activity or binding (including but not limited to potency and efficacy). Clustering of descriptors or compounds or mixtures using various distance measures and clustering methods applied to all possible metrics of compounds, mixtures and descriptors, such as Euclidean distance, Earth-Mover's distance, k-nearest neighbors and t-distributed stochastic neighbor embedding, can be used to visualize and validate associations between one or more ORs and one or more odor notes, as well as measurements and characteristics of any other property, including but not limited to chemical, physical, physicochemical, psychophysical, organoleptic, psychological, emotional, biological and composite properties.

[0093] Furthermore, common olfactory notes can be identified by comparing several sources of olfactory descriptions of compounds of interest, including, but not limited to, perfumer descriptions, flavorist descriptions, trained and untrained panelist descriptions, publicly available fragrance compound description databases, published flavor compound description databases, and F&F industry knowledge and expertise. As used herein, a "perfumer" is an expert in the field of fragrance who is able to distinguish and describe fragrance notes, whether singly or in combination.

[0094] In a further embodiment, odor notes linked to the activity of an OR can be identified by specifically testing the receptor with a) a set of compounds with a common odor note but different chemical structures, and b) a set of compounds with similar chemical structures (e.g., based on a structure-activity relationship (SAR) approach) but different odor notes. A comparison between the two sets of compounds with respect to receptor activation then reveals both the chemical requirements for agonism and the common odor notes of agonists for the receptor.

[0095] The methods according to the invention can also be used to characterize the specific odor notes of malodorous compounds. Well-characterized malodorous ORs can be screened for compounds that modulate, e.g., enhance, inhibit, or allosterically inhibit, the malodorous OR.

[0096] The methods presented herein are not limited to a particular class of OR, and include class I and class II ORs, receptors activated by flavors, and fragrance or malodorous compounds.

[0097] Methods for monitoring OR activity As long as the function of an OR is not impaired, it can be used in any form in the methods or assays described herein. For example, tissues or cells that endogenously express ORs, such as olfactory neurons isolated from living organisms and cultures thereof, olfactory cell membranes carrying ORs, recombinant cells genetically modified to express ORs and cultures thereof, membranes of recombinant cells, and artificial lipid bilayer membranes carrying ORs can be used.

[0098] Indicators for monitoring the activity of olfactory receptors include, for example, fluorescent calcium indicator dyes, calcium indicator proteins (e.g., GCaMP, a genetically encoded calcium indicator), fluorescent cAMP indicators, cell mobilization assays, cell dynamic mass redistribution assays, label-free cell-based assays, cAMP response element (CRE)-mediated reporter proteins, biochemical cAMP HTRF assays, β-arrestin assays, or electrophysiological recordings. In certain embodiments, calcium indicator dyes that can be used to monitor the activity of olfactory receptors expressed on the membranes of olfactory neurons are selected (e.g., Fura-2 AM). Compounds are sequentially screened, and odorant-dependent changes in calcium dye fluorescence can be measured using a fluorescence microscope or a fluorescence-activated cell sorter (FACS).

[0099] As an example, olfactory neurons activated by target agonists, e.g., compounds with specific olfactory notes, are isolated using either glass microelectrodes attached to a micromanipulator or a FACS machine. Mouse olfactory neurons are then analyzed by Ca ion spectroscopy, similar to previously described procedures. 2+Screening is performed by imaging (Malnic, B., et al. Cell 96, 713-723 (1999); Araneda, RC et al. J. Physiol. 555, 743-756 (2004); and International Publication No. 2014 / 210585). In particular, the use of a motorized, movable microscope stage increases the number of cells that can be screened to at least 1,500 per experiment. Because mice have approximately 1,200 different olfactory receptors and each olfactory neuron expresses only one of the 1,200 olfactory receptor genes, this screening capacity virtually covers the entire mouse odorant receptor repertoire. In other words, the combination of calcium imaging for high-throughput olfactory neuron screening leads to the identification of almost all odorant receptors that respond to a specific odorant profile. In one embodiment, odorant receptors that respond to target agonists, for example, those with a specific olfactory note, can be isolated for receptor identification. For example, at least one neuron is isolated for receptor identification.

[0100] Human or non-human mammalian receptors for target agonists, such as those with one or more specific olfactory notes, 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.The assays can be cell-based assays or binding assays, and the methods for identifying compounds can be high-throughput screening assays.More specifically, provided herein is a receptor-based assay that can be adapted to high-throughput screening of receptors using compound libraries to discover positive allosteric modulators, negative allosteric modulators, antagonists, or inverse agonist modulator compounds for specific agonists of interest.

[0101] In one embodiment, target agonist receptor gene sequences (e.g., having at least one olfactory note) are identified from target agonist-sensitive cells as follows: Pooled neurons are heated at 75°C for 10 minutes to disrupt cell membranes and make their mRNA available for amplification. This amplification step is important when applying NGS techniques with limited starting material, typically 1-15 cells. Multiple amplification protocols exist. For example, linear amplification by the Eberwine method (IVT) ensures that the relative transcription levels of expressed genes are maintained. To obtain sufficient amounts of cRNA, two consecutive overnight (14-hour) in vitro transcription rounds are used. The amplified cRNA is then used to generate an Illumina HiSeq cDNA library. The resulting short sequences (commonly referred to as "reads"), typically 75-150 base pairs long, are aligned to the mouse reference genome (such as UCSC version mm9 or mm10) to construct a complete transcriptome of these cells. Quantitative analysis of the transcriptome data yields a list of transcribed odorant receptor genes and their expression levels. The odorant receptor gene that shows the most abundant level of mRNA (most abundant "read") or is present in two or more replicate experiments is considered the putative target agonist receptor.

[0102] The predicted mouse OR genes are then used to mine mouse and human genome databases to identify the most closely related receptors (i.e., the most sequence similarity) in mice (paralogous genes) and humans (orthologous genes). This process can be performed using the BLAST search algorithm (publicly available on the NCBI website), a sequence similarity search tool, using all predicted gene sequences previously obtained from the initial transcriptome analysis as the query sequence. Newly identified genes from this data mining are considered potential receptors for specific olfactory notes, assuming that paralogous and orthologous genes are likely to have similar activities. In certain embodiments, pairwise comparisons of sequence homology are performed to identify closely related receptors in mice and humans, as described in WO 2014 / 210585. Other approaches, such as RT-PCR and microarray or mass spectrometry approaches, can also be used.

[0103] In a further embodiment, to complete the deorphanization process, candidate OR genes can be further expressed in vitro to confirm their activity against the compounds used in olfactory isolation, or their human orthologs identified as described in the previous embodiment, identified as responding to target agonists (e.g., having one or more specific olfactory notes), modified at their N-termini with short polypeptide sequences (e.g., FLAG® (SEQ ID NO: 2), Rho (SEQ ID NO: 4; the first 20 amino acids of the bovine rhodopsin receptor), and / or Lucy (SEQ ID NO: 6; a cleavable leucine-rich signal peptide sequence) tags, transiently expressed in HEK 293T cells, and separately stimulated with target agonists (e.g., having specific olfactory notes) to confirm their identity as bona fide receptors for specific target agonists. In a further embodiment, the RTP1 gene can also be expressed in cell lines, whether through activation of the endogenous RTP1 gene or through transformation or viral transduction, as described in WO2016201153. In this cell-based assay, the human Gα subunit Gαolf Co-expression of Gα15 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 Gα subunit Gα15 in this cell-based assay leads to an increase in internal Ca upon binding to an appropriate ligand. 2+ Activates the Gq transduction pathway, resulting in an increase

[0104] In a further embodiment, the compound is contacted with an OR, or a chimera or fragment thereof, wherein the OR, or a chimera or fragment thereof is expressed in a cell recombinantly modified to express the OR, or a chimera or fragment thereof.

[0105] In further embodiments, molecular 3D receptor modeling of ORs is used to assess binding potential in silico and identify compounds that may activate, mimic, block, inhibit, modulate, and / or enhance the activity of ORs. In further embodiments, machine learning algorithms known to those skilled in the art, such as support vector machines, random forests, XGBoost, graph convolutional networks, recurrent neural networks, variational autoencoders, generative adversarial networks, and the like, can be combined with OR activity data, molecular 3D receptor structure data, molecular 3D compound structure data, and physicochemical data to assess binding potential in silico and predict compounds that may activate, mimic, block, inhibit, modulate, and / or enhance the activity of ORs.

[0106] The activity of the compounds can be determined using in vivo, ex vivo, in vitro and synthetic screening systems.

[0107] In one embodiment, the contacting can be performed with a liposome or virus-induced budding membrane containing a polypeptide described herein.

[0108] In another embodiment, the methods for identifying compounds that bind to, suppress, block, inhibit, and / or modulate the activity of ORs may be performed on membrane fractions from intact cells or cells expressing the polypeptides described herein.

[0109] The ORs described herein can be used to identify modulatory compounds, such as inhibitors or antagonists, that will reduce the perception of specific olfactory notes associated with the OR.

[0110] In further embodiments, the use of antagonists against ORs encoding specific olfactory notes can be used to reveal other remaining notes in a compound or mixture of multiple compounds. It is known that the human ability to consciously and simultaneously attend to different olfactory notes is limited (e.g., Keller, A (2011)). When smelling a multi-note odor or scent, attention shifts between notes, so that attention can be focused on only one note at a time. While not wishing to be limited to a particular theory of attentional modulation, suppression of the note encoded by an antagonized OR would remove it from the pool of notes that inevitably compete for attention, allowing the remaining notes to occupy attentional space instead. In some cases, a relative, rather than absolute, change in the activity of the remaining ORs leads to an apparent perceptual enhancement of the note they encode.

[0111] Polypeptides applicable according to the present invention The present invention includes functional equivalents or analogs or functional variants of the OR polypeptides specifically described herein.

[0112] A functional equivalent refers to a polypeptide that exhibits at least 1-10%, or at least 20%, or at least 50%, or at least 75%, or at least 90%, or at least 95% greater or less OR activity in the assay used for OR activity.

[0113] According to the present invention, functional equivalents also encompass specific variants of the amino acid sequences described herein, which have, at at least one sequence position, an amino acid different from those specifically mentioned, but which still possess one of the aforementioned biological activities. Thus, functional equivalents include variants obtained by addition, substitution, in particular conservative substitution, deletion and / or inversion of one or more, for example 1-20, 1-15 or 5-10, amino acids, where the mentioned changes can occur at any sequence position, provided that they result in a variant with a profile of properties according to the invention. Functional equivalence is particularly relevant when the activity pattern qualitatively coincides between the variant and the unchanged polypeptide, i.e., for example, interaction with the same agonist or antagonist, but at different rates (i.e., EC 50 Or IC 50 Examples of suitable (conservative) amino acid substitutions are provided in the table below. [Table 1]

[0114] Functional equivalents in the above sense also include precursors of the described polypeptides, as well as functional derivatives and salts of the polypeptides. Precursors are natural or synthetic precursors of the polypeptides, with or without the desired biological activity.

[0115] The term "salts" refers not only to salts of carboxyl groups but also to acid addition salts of amino groups of the protein molecules according to the present invention. Salts of carboxyl groups can be prepared by known methods and include inorganic salts, such as sodium salts, calcium salts, ammonium salts, iron salts and zinc salts, as well as salts with organic bases, such as amines such as triethanolamine, arginine, lysine, piperidine, etc. The present invention also includes acid addition salts, such as salts with inorganic acids such as hydrochloric acid and sulfuric acid, and salts with organic acids such as acetic acid and oxalic acid.

[0116] Functional derivatives of the polypeptides of the invention can be produced at functional amino acid side groups or at their N- or C-termini using known techniques, including, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtained by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups formed by reaction with acyl groups, or O-acyl derivatives of free hydroxyl groups formed by reaction with acyl groups.

[0117] Functional equivalents also include polypeptides obtained from other organisms and naturally occurring variants. For example, regions of homologous sequence regions can be established by sequence comparison, and equivalent polypeptides can be determined based on the specific parameters of the invention.

[0118] Functional equivalents also include, for example, fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention which exhibit a desired biological function.

[0119] Functional equivalents include fusion proteins having one of the polypeptide sequences described herein or a functional equivalent derived therefrom and at least one additional, functionally distinct heterologous sequence in N- or C-terminal association (i.e., without substantial mutual dysfunction of the fusion protein moieties). Non-limiting examples of these heterologous sequences include, for example, a signal peptide, a histidine anchor, or an enzyme.

[0120] Functional equivalents according to the present invention include homologs of the specifically disclosed polypeptides, which have a homology (or identity) of at least 60%, preferably at least 75%, in particular at least 80 or 85%, such as 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, to one of the specifically disclosed amino acid sequences, as calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85(8), 1988, 2444-2448. Percent homology or identity of homologous polypeptides according to the present invention refers in particular to the identity, expressed in percentage, of amino acid residues based on the full length of one of the amino acid sequences specifically described herein.

[0121] Identity data expressed as a percentage can also be determined with the help of BLAST alignment, the algorithm blastp (protein-protein BLAST) or by applying the Clustal settings specified herein below.

[0122] In the case of possible protein glycosylation, functional equivalents according to the present invention include the polypeptides described herein in deglycosylated or glycosylated forms, as well as modified forms that can be obtained by altering the glycosylation pattern.

[0123] Such functional equivalents or homologues of the polypeptides according to the invention can be generated by mutagenesis, for example by point mutation, lengthening or shortening of the protein, or as described in more detail below.

[0124] Functional equivalents or homologs of the polypeptides of the present invention can be identified by screening combinatorial databases of variants, such as truncation variants. For example, a diverse database of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, e.g., enzymatic ligation of a mixture of synthetic oligonucleotides. Numerous methods can be used to generate a database of potential homologs from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed using an automated DNA synthesizer, and the synthetic genes can then be ligated into an appropriate expression vector. Using degenerate genes, all sequences in the mixture that encode a set of desired candidate protein sequences can be ligated. Methods for synthesizing degenerate oligonucleotides are known to those skilled in the art (e.g., Narang, SA (1983); Itakura et al. (1984) (a); Itakura et al., (1984) (b); Ike et al. (1983)). This method also includes the generation of codon-optimized nucleic acid sequences, i.e., polypeptides adapted to the codon usage of the host cell.

[0125] Several techniques are known for screening gene products from combinatorial databases created by point mutation or truncation, and for screening cDNA libraries to obtain gene products with selected properties. These techniques can be adapted for rapid screening of gene banks created by combinatorial mutagenesis of homologs according to the present invention. The most frequently used technique for screening large gene banks based on high-throughput analysis involves cloning the gene bank in replicable expression vectors, transforming appropriate cells with the resulting vector database, and expressing the combinatorial genes under conditions that facilitate the isolation of vectors encoding genes whose products are detected by detecting the desired activity. Recursive Ensemble Mutagenesis (REM), a technique that increases the frequency of functional variants in databases, can be used in conjunction with screening tests to identify homologs (Arkin and Yourvan (1992); Delgrave et al. (1993)).

[0126] Nucleic acid sequences according to the present invention The present invention includes nucleic acid sequences that encode the polypeptides of the present invention.

[0127] The present invention also relates to nucleic acids that have a degree of "identity" to the sequences specifically disclosed herein. "Identity" between two nucleic acids in each case refers to nucleotide identity over the entire length of the nucleic acid.

[0128] For example, identity can be calculated using the Vector NTI Suite 7.1 program (Higgins DG, Sharp PM. ((1989))) from Informax (USA) employing the Clustal method, with the following settings: Multiple alignment parameters Gap Opening Penalty 10 Gap Extension Penalty 10 Gap Separation Penalty Range 8 Gap Separation Penalty Off % identity for alignment delay 40 Residue specificity gap off Hydrophilic residue gap off Transition weighting: 0 Pairwise alignment parameters: FAST Algorithm On K tuple size 1 Gap Penalty 3 Window Size 5 Best diagonal number 5

[0129] Alternatively, identity may be determined according to Chenna, et al. (2003), web page: http: / / www.ebi.ac.uk / Tools / clustalw / index.html#, and the following settings: DNA Gap Open Penalty 15.0 DNA gap extension penalty 6.66 DNA Matrix Identity Protein Gap Open Penalty 10.0 Protein extension penalty 0.2 Protein Matrix Gonnet Protein / DNA ENDGAP -1 Protein / DNA GAPDIST 4

[0130] The present invention also relates to nucleic acid sequences (single- and double-stranded DNA and RNA sequences, e.g., cDNA and mRNA) encoding one of the above-mentioned polypeptides and functional equivalents thereof, which may be obtained, for example, using artificial nucleotide analogues.

[0131] The present invention relates to both isolated nucleic acid molecules encoding a polypeptide according to the invention, or a biologically active segment thereof, and to nucleic acid fragments that can be used, for example, as hybridization probes or primers to identify or amplify the encoding nucleic acid according to the invention.

[0132] Nucleic acid molecules according to the invention may further comprise untranslated sequences from the 3' and / or 5' ends of the coding gene region. The present invention further relates to nucleic acid molecules that are complementary to the specifically described nucleotide sequences or segments thereof.

[0133] The nucleotide sequences according to the invention allow the generation of probes and primers that can be used to identify and / or clone homologous sequences in other cell types and organisms. A probe or primer generally comprises a nucleotide sequence region that hybridizes under "stringent" conditions (see below) over at least about 12, preferably at least about 25, e.g., about 40, 50 or 75 consecutive nucleotides of the sense strand or the corresponding antisense strand of a nucleic acid sequence according to the invention.

[0134] An "isolated" nucleic acid molecule is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid, and further can be substantially free of other cellular material or culture medium if it is produced by recombinant techniques, or free of chemical precursors or other chemicals if it is chemically synthesized.

[0135] "Hybridize" refers to the ability of a polynucleotide or oligonucleotide to bind to a nearly complementary sequence under standard conditions, but under these conditions nonspecific binding between non-complementary partners does not occur. For this reason, sequences can be 90-100% complementary. The property of complementary sequences to be able to bind specifically to each other is utilized, for example, in Northern or Southern blotting, or in primer binding in PCR or RT-PCR.

[0136] Short oligonucleotides of the conserved region are advantageously used for hybridization.However, it is also possible to use longer fragments or complete sequences of the nucleic acid according to the present invention for hybridization.These "standard conditions" vary depending on the nucleic acid (oligonucleotide, longer fragment or complete sequence) used or the type of nucleic acid (DNA or RNA) used for hybridization.For example, the melting temperature of DNA:DNA hybrid is about 10°C lower than that of DNA:RNA hybrid of the same length.

[0137] For example, depending on the specific nucleic acid, standard conditions refer to a temperature of 42-58°C in a buffer solution of 0.1-5xSSC (1xSSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2), or even in the presence of 50% formamide, e.g., 42°C in 5xSSC, 50% formamide. Advantageously, hybridization conditions for DNA:DNA hybrids are 0.1xSSC, with temperatures of about 20-45°C, preferably about 30-45°C. For DNA:RNA hybrids, hybridization conditions are advantageously 0.1xSSC, with temperatures of about 30-55°C, preferably about 45-55°C. These stated temperatures for hybridization are examples of melting temperature values ​​calculated for a nucleic acid having a length of about 100 nucleotides and a 50% G+C content in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant genetic textbooks, such as Sambrook et al., 1989, and can be calculated using formulas known to those skilled in the art, depending on, for example, the length of the nucleic acid, the type of hybrid, or the G+C content. Those skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), (1985), Brown (ed) (1991).

[0138] "Hybridization" can be carried out under particularly stringent conditions, such as those described in Sambrook (1989) or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

[0139] As used herein, the term hybridization or hybridizing under certain conditions is intended to describe hybridization and washing conditions under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions may be such that sequences with at least about 70%, for example, at least about 80%, for example, at least about 85%, 90%, or 95% identity remain bound to each other.

[0140] Appropriate hybridization conditions can be selected by one of ordinary skill in the art with minimal experimentation, as exemplified by Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Further stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0141] The defined conditions for low stringency are as follows: DNA-containing filters are pretreated for 6 hours at 40°C in a solution containing 35% formamide, 5x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (w / v) dextran sulfate, and 5–20x10 6 Use 32P-labeled probe. Filters are incubated in hybridization mixture at 40°C for 18-20 hours, then washed at 55°C for 1.5 hours in a solution containing 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated at 60°C for an additional 1.5 hours. Filters are blotted dry and exposed for autoradiography.

[0142] The defined conditions for medium stringency are as follows: DNA-containing filters are pretreated for 7 hours at 50°C in a solution containing 35% formamide, 5x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (w / v) dextran sulfate, and 5–20x10 6 32P-labeled probe is used. Filters are incubated in hybridization mixture at 50°C for 30 hours, then washed at 55°C for 1.5 hours in a solution containing 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated at 60°C for an additional 1.5 hours. Filters are blotted dry and exposed for autoradiography.

[0143] The defined conditions for high stringency are as follows: Prehybridization of DNA-containing filters is performed for 8 hours to overnight at 65°C in a buffer consisting of 6x SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg / ml denatured salmon sperm DNA; 100 μg / ml denatured salmon sperm DNA and 5–20 × 10 6 The filters were hybridized in a prehybridization mixture containing 100 cpm of P-labeled probe at 65°C for 48 hours. The filters were washed in a solution containing 2x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA at 37°C for 1 hour, followed by a 45-minute wash in 0.1x SSC at 50°C.

[0144] Other conditions of low, medium, and high stringency well known in the art (e.g., conditions used for cross-species hybridizations) can be used when the above conditions are inappropriate (e.g., conditions used for cross-species hybridizations).

[0145] Nucleic acid sequences according to the invention can be derived from the sequences specifically disclosed herein and can differ therefrom by the addition, substitution, insertion or deletion of individual or multiple nucleotides, and can further encode polypeptides having a desired profile of properties.

[0146] The present invention also encompasses nucleic acid sequences which contain so-called silent mutations or which have been altered in accordance with the codon usage of a particular origin or host organism compared to the specifically described sequences, as well as naturally occurring variants thereof, such as splice variants or allelic variants.

[0147] Derivatives of nucleic acid sequences according to the present invention refer, for example, to allelic variants having at least 60% homology at the derived amino acid level, preferably at least 80% homology, particularly preferably at least 90% homology over the entire sequence range (with regard to homology at the amino acid level, see the details above for polypeptides). Advantageously, the homology may be higher over subregions of the sequence.

[0148] Furthermore, derivatives should also be understood as homologs of the nucleic acid sequences according to the invention, such as animal, plant, fungal or bacterial homologs, truncated sequences, single-stranded DNA or RNA coding and non-coding DNA sequences, for example, homologs which, at the DNA level, have at least 40%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80% homology over the entire DNA region given in the sequences specifically disclosed herein.

[0149] Furthermore, derivatives should be understood to be, for example, fusions with promoters.The promoters added to the described nucleotide sequences can be modified by at least one nucleotide exchange, at least one insertion, inversion and / or deletion, but the functionality or effectiveness of the promoters is not impaired.Furthermore, the effectiveness of promoters can be improved by changing their sequences, or even in organisms of different genera, they can be completely replaced with more effective promoters.

[0150] Furthermore, those skilled in the art are familiar with methods for generating nucleotide sequences encoding functional variants, i.e., polypeptides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 2, 4, 6, or 8, and / or polypeptides encoded by nucleic acid molecules comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NOs: 1, 2, 5, or 7.

[0151] Depending on the technique used, one skilled in the art can introduce completely random or more directed mutations into genes or non-coding nucleic acid regions (such regions being important for regulating expression, for example) and then generate gene libraries. The molecular biology methods required for this purpose are known to those skilled in the art and are described, for example, in Sambrook and Russell, Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001.

[0152] Methods for modifying genes, and thus the polypeptides encoded by them, have long been known to those skilled in the art, and include, for example: - site-directed mutagenesis, in which individual or multiple nucleotides of a gene are replaced in a directed manner (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey), - saturation mutagenesis, which allows exchanging or adding codons for any amino acid at any point in a gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22:1593; Barettino D, Feigenbutz M, Valcarel R, Stunnenberg HG (1994) Nucleic Acids Res 22:541; Barik S (1995) Mol Biotechnol 3:1), - error-prone polymerase chain reaction, in which the nucleotide sequence is mutated by an error-prone DNA polymerase (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18:3739), - The SeSaM method (sequence saturation method), in which favorable exchange is prevented by the polymerase. Schenk et al., Biospektrum, Vol. 3, 2006, 277-279. - passage of genes in mutator strains, where an increased mutation rate of nucleotide sequences occurs due to defects in DNA repair mechanisms (Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E. coli mutator strain. In: Trower MK (Ed.) In vitro mutagenesis protocols. Humana Press, New Jersey), or - DNA shuffling, in which a pool of closely related genes is formed, digested, and the fragments are used as templates for polymerase chain reaction, ultimately generating a full-length mosaic gene by repeated strand separation and reannealing (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747).

[0153] Using so-called directed evolution (as described, inter alia, in Reetz MT and Jaeger KE (1999), Topics Curr Chem 200:31; Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods for optimizing industrial polypeptides by directed evolution, In: Demain AL, Davies JE (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), those skilled in the art can generate functional variants in a directed manner on a large scale. For this purpose, in a first step, a gene library for each polypeptide is first generated, for example, using the methods indicated above. This gene library is then expressed in a suitable manner, for example, by means of a bacterial or phage display system.

[0154] The relevant gene of the host organism that expresses functional mutants with properties roughly corresponding to the desired properties can be subjected to another mutation cycle. The mutation and selection or screening steps can be repeated repeatedly until the functional mutants present have the desired properties to a sufficient degree. Using this iterative procedure, a limited number of mutations, for example, 1, 2, 3, 4 or 5 mutations, can be carried out stepwise, and their effects on the target activity can be evaluated and selected. The selected mutants can then be subjected to further mutation steps in the same manner. In this way, the number of individual mutants to be investigated can be significantly reduced.

[0155] The results according to the present invention also provide important information regarding the structure and sequence of related polypeptides that is required for the targeted generation of further polypeptides with desired modified properties. In particular, it is possible to define so-called "hot spots", i.e., sequence segments that may be suitable for modifying properties by introducing targeted mutations.

[0156] It is also possible to derive information regarding positions in the amino acid sequence where mutations that are expected to have little effect on activity may be made nearby; such mutations may be referred to as potential "silent mutations."

[0157] For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector that allows optimal expression of the gene in the host. Vectors are well known to those skilled in the art and can be found, for example, in "cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0158] The nucleic acid and amino acid sequences identified and / or used herein are set forth below: FLAG® SEQ ID NO:1-DNA [ka] SEQ ID NO:2 - Protein DYKDDDDK Rho tag SEQ ID NO:3-DNA [ka] SEQ ID NO:4 - Protein [ka] Lucy Tag SEQ ID NO:5-DNA [ka] SEQ ID NO:6 - Protein [ka] motif SEQ ID NO:7 MAYDRYVAIC motif SEQ ID NO:8 FSTCSSH motif SEQ ID NO:9 PMLNPFIY OR11A1 SEQ ID NO: 10-DNA [ka] SEQ ID NO: 11 - Protein [ka] OR8B3 SEQ ID NO: 12-DNA [ka] SEQ ID NO: 13 - Protein [ka] OR5AU1 SEQ ID NO: 14-DNA [ka] SEQ ID NO: 15 - Protein [ka] OR8D1 SEQ ID NO: 16-DNA [ka] SEQ ID NO: 17 - Protein [ka] OR5AN1 SEQ ID NO: 18-DNA [ka] SEQ ID NO: 19-DNA [ka] OR1N2 [ka] SEQ ID NO:21 - Protein [ka] OR5A1 SEQ ID NO: 22-DNA [ka] SEQ ID NO:23 - Protein [ka] OR7A17 SEQ ID NO: 24-DNA [ka] SEQ ID NO: 25 - Protein [ka] OR10J5 SEQ ID NO: 26-DNA [ka] SEQ ID NO: 27 - Protein [ka] OR1C1 SEQ ID NO: 28-DNA [ka] SEQ ID NO: 29 - Protein [ka] OR5B12 SEQ ID NO: 30 - DNA [ka] SEQ ID NO:31 - Protein [ka]

[0159] The following examples are illustrative and are not meant to limit the scope of the invention described in the abstract, specification, or claims.

[0160] Example The perfume notes described in the following examples have been provided by one or more perfumers.

[0161] Example 1: Olfactory quality coding by the peripheral olfactory system The data presented below are summarized in Figures 1 and 2 to conceptualize how olfactory notes are encoded at the periphery of the olfactory system. Figure 1 shows a fragrance wheel representing fragrance notes and notes and the relationship between the corresponding olfactory groups. Volatile compounds that exhibit specific olfactory notes on the fragrance wheel were mapped by the receptors they activate. Molecules that share a note activate the same receptor, regardless of chemical similarity. Molecules that share several olfactory notes consequently coactivate the same corresponding ORs. For example, molecules 1, 2, and 3 each activate two characterized receptors, exhibiting the two corresponding olfactory notes (shown in Figure 1).

[0162] This is further illustrated in Figure 2. Figure 2 shows the receptive fields of three receptors—OR5AU1 (SEQ ID NO: 15), OR8D1 (SEQ ID NO: 17), and OR8B3 (SEQ ID NO: 13)—each linked to a different olfactory note: lactonic coconut, fenugreek, or coumarin. Here, fenugreek is captured by the semantically related descriptors fenugreek, maple, and celery, and coumarin is captured by coumarin, tonka, and hay. Molecules exhibiting only one of these notes will correspondingly activate only one receptor. Molecules exhibiting a combination of these descriptors will activate the corresponding combination of receptors. Compounds at the intersection of two or more OR receptive fields will elicit two or more corresponding notes (Figure 2). As a result, the overall receptor activation profile (i.e., which set of receptors is activated) directly correlates with the sensory attributes of a given compound. Table 1 lists the notes of a subset of compounds located next to the receptors shown in Figure 2. Additional descriptors not represented by OR activity suggest that deorphanization and characterization of additional ORs remains to be determined. Note that the data presented in this example is not intended to be exhaustive. Many more olfactory notes and many more OR-odorant pairs exist beyond those represented in this fragrance wheel. However, the predictive aspects of OR screening become apparent in light of the compiled results. [Table 2]

[0163] Example 2: OR11A1 activity captures a single common organoleptic note: earthy. The molecular acceptance range of the human odorant receptor OR11A1 (SEQ ID NO: 11) was tested by performing a large-scale screening with a chemically and organoleptically diverse volatile compound library containing approximately 800 compounds. Using a cell-based assay, OR11A1 was tested in the HEK293T cell line, in which the endogenous RTP1 gene was activated and an odorant receptor chaperone was expressed (WO 2016 / 201153). The cells were transfected with Flag-Rho-tagged receptors and the canonical G protein G. olf The cells were co-injected with α-glucan and exposed to a single concentration of each test compound tested individually. Receptor activity was detected by measuring the increase in cytoplasmic cAMP using a homogeneous time-resolved fluorescence (HTRF)-based kit (CisBio, cAMP dynamic 2 kit, 62AM4PEJ).

[0164] First, a 300 mM stock solution of the 798 compounds was prepared in pure DMSO. The stock solution of each compound was further diluted to a final test concentration of 300 μM. The final DMSO concentration was 0.1%, which had no visible effect on the cells. Each compound was presented to a cell line expressing the olfactory receptor OR11A1. The quality of this high-throughput screening (HTS) process was determined by calculating the Z' value for each plate, assessing window variability and signal reliability (the average Z' value per plate was 0.72 ± 0.12, well within the required quality criteria of 0.5–1). The results of this single-concentration activation screen are shown in Figure 3, with hit compounds highlighted by light gray dots.

[0165] Subsequently, using the same cell-based assay as described above, dose-response experiments were performed on a subset of the best hit compounds, as shown in Figure 4, to confirm that the candidate hit compounds were true agonists (activators). A negative compound from the initial screening step (2,2,6,6-tetramethyl-1-cyclohexanone) was used as a negative control for response specificity, but no significant dose-response was obtained. Sensitivity (potency) and activation strength (efficacy) were measured using the EC50 The curves were calculated as a measure of the α (concentration required to reach half-maximal activation level) and Span (assay window span between baseline activity level and activation saturation plateau). These values ​​were used to represent the molecular acceptance range of OR11A1 by potency (X-axis) and efficacy (Y-axis) in Figure 5. The corresponding chemical structures are shown in Figure 6. The partial agonist, tonalide, is also listed. The five agonists cover different fragrance notes: woody (patchouli alcohol), musky (vulcanolide, tonalide), and camphoric (fenchyl alcohol) (Table 2). Collectively, this data demonstrates the diversity of both the organoleptic notes and chemical structures of compounds capable of activating OR11A1, consistent with the results described in WO 2016 / 201152 (musk) and EP 2 832 347 (patchouli). However, OR11A1 activity was specifically associated with the earthy facets of musk (WO 2016 / 201152) or patchouli (EP 2832347), rather than their odor notes. Indeed, the single common denominator among these compounds is earthiness, which is semantically captured by the following descriptors: earthy, humus, and earthy moss. These compounds are neither all musk nor all patchouli, but all are earthy. On the other hand, tonalide, a musk structurally related to vulcanolide but only a weak (partial) agonist of OR11A1, did not deliver a strong earthy note, despite its chemical and overall organoleptic similarity to vulcanolide (i.e., musky, powdery), as shown in Table 2. [Table 3]

[0166] Example 3: Odorant receptors encode specific organoleptic scent notes Following the same approach as described in Example 2, we applied a systematic screening of multiple Lucy-Flag-Rho-tagged human ORs to search for a link between receptor activity and the specific olfactory notes they encode. Analysis of the molecular acceptance range of ORs indicates that, although agonists may differ in their physicochemical properties (e.g., molecular weight, volatility, functional groups, three-dimensional structure, etc.) and their overall organoleptic notes, common specific olfactory notes can be identified in all cases. As a result, the following receptors are assigned to specific olfactory notes and are considered fundamental in eliciting the perception of these notes:

[0167] OR8B3, SEQ ID NO: 13, was activated by compounds that yield coumarin-like compounds (Figure 7, Table 3). [Table 4]

[0168] OR5AU1, SEQ ID NO: 15, was activated by compounds that produce lactonic coconut flavors (Figure 8, Table 4). [Table 5]

[0169] OR8D1, SEQ ID NO: 17, was activated by compounds that produce fenugreek notes (Figure 9, Table 5). [Table 6]

[0170] OR5AN1, SEQ ID NO: 19, is activated by structurally different molecules such as nitromusks and macrocyclic ketones to produce powdery musk notes commonly used in perfumery (Figure 10, Table 6). [Table 7]

[0171] OR1N2, SEQ ID NO: 21, is activated by large macrocycles (ketones and lactones) and produces an animalic musk note commonly used in perfumery (Figure 11, Table 7). [Table 8]

[0172] OR5A1, SEQ ID NO: 23, was activated by compounds that produced violet tones (Figure 12, Table 8). [Table 9]

[0173] OR7A17, SEQ ID NO: 25, was activated by compounds that produce blond wood (also described as creamy wood) notes (Figure 13, Table 9). [Table 10]

[0174] OR10J5, SEQ ID NO: 27, was activated by compounds that produce a lily of the valley tone (Figure 14, Table 10). [Table 11]

[0175] OR1C1, SEQ ID NO: 29, was activated by compounds that produce linalic tones (Figure 15, Table 11). [Table 12]

[0176] OR5B12, SEQ ID NO: 31, was activated by compounds that produce a jasmine note (Figure 16, Table 12). [Table 13]

[0177] Example 4: Using odorant receptor activity to analyze and generate complex mixtures Overall odorant receptor activity is used to determine the scent of a composition containing multiple volatile molecules. The activity of a composition on an ensemble of ORs reflects the final activity achieved by the composition on each OR, taking into account OR modulation (e.g., inhibition and enhancement) that may occur when the ORs are contacted by multiple compounds. The resulting activity profile is used to describe the overall scent notes of the composition, establishing a link between OR activity and perceived scent notes. Alternatively, the activity profile required for a target scent can be inferred and used as a signature activity for recreating the target scent under different conditions. Similarly, such a signature activity profile can be used as a quality assessment or quality benchmark test.

[0178] The activity of a composition can also be used as a feature to guide the creation of new compositions that replicate the activity of the original composition. Such new compositions can be obtained by modifying the initial composition while retaining the original ensemble of activated ORs. Such modifications can include removing irrelevant compounds, replacing a compound with a preferred compound, replacing one compound with multiple preferred compounds, replacing multiple compounds with one compound that fulfills the same role as the replaced compounds, or replacing multiple compounds with multiple different compounds. New compositions with similar or identical activity to the target composition will have similar odor characteristics.

[0179] Finally, the compounds required to achieve the intended scent are estimated and a new composition is created. For example, a composition with a desired set of scent notes is created. Alternatively, a composition can be designed to eliminate one or more undesirable scent notes, such as malodors. A composition can also be created by simultaneously employing both strategies.

[0180] Example 5: Modeling OR activity of complex mixtures can lead to prediction of perceptible odor notes The fragrance accord (Accord A) contained ingredients that activate the odorant receptors OR8B3 (resulting in a hay note), OR8D1 (resulting in a celery note), and OR5AU1 (resulting in a lactonic coconut note), along with three other accords with minor modifications (Accords B-D). See Table 12. [Table 14]

[0181] Ingredients that did not activate any of the three target ORs were aggregated and listed as "other ingredients" for simplicity. Receptor screening data (described in Example 1) was used for each OR to identify alternative activators that could be used in place of the ingredients present in the original formula. This approach allowed us to prioritize the use of preferred ingredients due to the optimization of multiple factors, including reduced cost, increased biodegradability, or the use of compounds with environmentally friendly properties. The overall activity of the Accords against each receptor can be calculated using a competitive binding model by considering not only the component ratios in the mixture, as shown in Table 12, but also the individual activity levels against each receptor, as shown in Figure 17. Activity levels were normalized to the maximum cellular response to forskolin, a known pharmacological transduction cascade activator, which served as an anchor point for data comparison. Accord activity predicted by the model was compared to cell-based data generated using the same Accords (cell-based assay described in Example 2). Consistent activity levels were observed between the model and in vitro data, validating the model. The resulting activity predictions between the original and modified accords were used to make sensory predictions based on the OR activity change (i.e., more or less active) and to infer the corresponding change in odor notes (i.e., stronger or weaker). The sensory predictions were validated by a sensory panel of fragrance experts, who performed blind evaluations of three target odor notes for each accord: hay, celery, and lactonic coconut.

[0182] The activity of OR8D1 is demonstrated by comparing Accords A and B, where cyclotone was removed and replaced with maple furanone. The target activity level was set to lower OR8D1 relative to the original activity level. Figure 18 shows the predicted activity levels from the model compared to the in vitro receptor validation, normalized to a 100% forskolin response. A corresponding decrease in the perception of a celery note was predicted, which was confirmed by expert panel evaluation, as summarized in Figure 19.

[0183] The activity of OR8B3 and OR5AU1 is demonstrated by comparing Accords A and C, where nonalactone was removed and replaced with coumarin. In this case, the target activity level was set to be high for OR8B3 and null for OR5AU1 relative to the original activity level. Figure 20 shows the predicted activity levels from the model compared to in vitro validation of both receptors, normalized to a 100% forskolin response. A corresponding increase in the perception of 'hey' and loss of lactonic coconut notes was predicted, which was confirmed by expert panel evaluation, as summarized in Figure 21.

[0184] The activity of OR8B3 and OR5AU1 is demonstrated by comparing Accords A, C, and D, which remove nonalactone, replace it with coumarin, and add tuberolide. In this case, the target activity level was set to leave OR8B3 unchanged relative to Accord C, while restoring OR5AU1 to its original activity level. Figure 20 shows the predicted activity levels from the model compared to in vitro validation of both receptors, normalized to a 100% forskolin response. A corresponding restoration of the coconut-like perception was predicted, which was confirmed by expert panel evaluation, as summarized in Figure 22.

[0185] These examples of fragrance accord modification capture the system's ability to model receptor activity of complex mixtures before and after compound removal and replacement using only single compound input data (such as those obtained in Examples 1-3). The system's ability to predict sensory outcomes based on receptor activity calculations was also demonstrated by maintaining the perception of specific desired odor notes with different compounds, adjusting the concentration or ratio of replacement compounds in the mixture to decrease or increase the desired odor notes, and rebalancing the composition by adding compounds to restore desired odor notes lost during modification.

[0186] The contents of all patents, publications, and published patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for associating at least one olfactory note with an olfactory receptor, comprising: (a) providing an olfactory receptor that is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to 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, or SEQ ID NO:31; (b) contacting the olfactory receptor with a compound having at least one known odor note; (c) determining whether the compound activates the olfactory receptor; (d) repeating steps (b) and (c) with a compound having at least one known odor note, wherein said compound in step (d) is different from said compound in steps (b) and (c) of the preceding iteration; (e) classifying the compounds from steps (b)-(d) that activate the olfactory receptor into a subset; (f) identifying at least one olfactory note common to said subset of compounds; (g) assigning the identified at least one odor note to the olfactory receptor; A method comprising:

2. 1. A method for screening at least one compound having a particular odor note, comprising: (a) providing an olfactory receptor that is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to 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, or SEQ ID NO:31, associated with the at least one identified odor note of claim 1; (b) contacting the olfactory receptor with at least one compound; (c) determining whether the at least one compound activates the olfactory receptor; (d) associating said at least one compound with said identified at least one olfactory note if said at least one compound activates said olfactory receptor; A method comprising:

3. 3. The method of claim 2, wherein a plurality of olfactory receptors, each having at least one different identified odor note, is provided in step (a), and the plurality of identified odor notes are associated in step (d) with a compound or combination of compounds that activates the plurality of olfactory receptors according to steps (b) and (c).

4. 1. A method for screening at least one compound for an arthy, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating an ARTIFICIAL SYNTHESIS with said at least one compound if said at least one compound activates said polypeptide; wherein the polypeptide is an olfactory receptor.

5. 1. A method for screening at least one compound for coumarin activity, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating a coumarin moiety with said at least one compound if said at least one compound activates said polypeptide; wherein the polypeptide is an olfactory receptor.

6. 1. A method for screening at least one compound for lactonic coconut flavor, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating a lactonic coconut flavor with said at least one compound if said at least one compound activates said polypeptide; wherein the polypeptide is an olfactory receptor.

7. 1. A method for screening at least one compound for fenugreek flavor, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating a fenugreek flavor with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

8. 1. A method for screening at least one compound for a powdery musk note, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating a powdery musk note with said at least one compound if said at least one compound activates said polypeptide; wherein the polypeptide is an olfactory receptor.

9. 1. A method for screening at least one compound for an animal musk note, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating an animalic musk note with said at least one compound if said at least one compound activates said polypeptide; wherein the polypeptide is an olfactory receptor.

10. A method for screening at least one compound for violet hue is provided, the method comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating a violet hue with said at least one compound if said at least one compound activates said polypeptide; wherein the polypeptide is an olfactory receptor.

11. 1. A method for screening at least one compound for blond wood tone, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating a blond wood odor note with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

12. 1. A method for screening at least one compound for lily of the valley tone, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:27; (b) contacting said polypeptide with said at least one compound; (c) determining whether said at least one compound activates said polypeptide; (d) associating a lily of the valley tone with the at least one compound if the at least one compound activates the polypeptide; wherein the polypeptide is an olfactory receptor.

13. 1. A method for screening at least one compound for linaloic acid, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:29; (b) contacting said polypeptide with said at least one compound; (c) determining whether the compound or combination of compounds activates the polypeptide; (d) associating a linalic acid with said at least one compound if said at least one compound activates said polypeptide; wherein the polypeptide is an olfactory receptor.

14. 1. A method for screening at least one compound for jasmine notes, comprising: (a) providing a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31; (b) contacting said polypeptide with said at least one compound; (c) determining whether the compound or combination of compounds activates the polypeptide; (d) associating a jasmine note with said at least one compound if said at least one compound activates said polypeptide; wherein the polypeptide is an olfactory receptor.

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