Methods of determining susceptibility of olfactory receptor ligands to adaptation

Abstract

The present disclosure generally provides methods for identifying olfactory receptor ligands that are more or less likely to be susceptible to olfactory adaptation, and the cellular assays related to identifying such ligands. In certain embodiments, the methods comprise determining a degree to which certain receptors are internalized by a cell following contact with a receptor ligand. In some embodiments, the methods comprise identifying which ligands are more susceptible or less susceptible to olfactory adaptation based on the determined degree of internalization.

Description

[0001] METHODS OF DETERMINING SUSCEPTIBILITY OF OLFACTORY RECEPTOR LIGANDS TO ADAPTATION

[0002] TECHNICAL FIELD

[0003] The present disclosure generally provides methods for identifying olfactory receptor ligands that are more or less likely to be susceptible to olfactory adaptation, and the cellular assays related to identifying such ligands. In some embodiments, the ligand is an olfactory receptor agonist, and, in other embodiments, the ligand is an olfactory receptor antagonist. In certain embodiments, the methods comprise determining a degree to which certain receptors are internalized by a cell following contact with a volatile compound. In some embodiments, the methods comprise identifying which agonists are more susceptible or less susceptible to olfactory adaptation based on the determined degree of internalization.

[0004] DESCRIPTION OF RELATED ART

[0005] People commonly experience the physiological phenomenon of olfactory adaptation. Certain fragrances or odors seem to linger, and one continues to smell them as long as these fragrances or odors are present. But other fragrances or odors seem not to linger, even though they remain present. Thus, humans tend to adapt to certain fragrances and odors but not to others. Adaptation thus results in a decreased perceived intensity of volatile odorants and mixtures thereof over prolonged exposure.

[0006] There has been no singular explanation for such adaptation, or the lack of it. Some have suggested that it is of psychological nature. After all, some people tend to experience olfactory adaptation in situations where others do not. But it has also been suggested that there may be a molecular or cellular explanation as well. A variety of different biochemical mechanisms for adaptation have been proposed.

[0007] There may be many benefits to identifying a biochemical explanation, as it would permit the rapid and efficient identification of compounds that may be susceptible to adaptation and therefore as less useful as fragrances in situations where a long-term olfactory stimulation is desired or more useful in situations where a long-term olfactory stimulation is not desired. Additionally, it would also permit the rapid and efficient identification of compounds that may be less susceptible to adaptation and therefore more useful as fragrances in situations where a long-term olfactory stimulation is desired or less useful as fragrances in situations where a longterm olfactory stimulation is not desired. Otherwise, one is left to test for olfactory adaptation through human sensory testing, which can be time-intensive, expensive, and inefficient.

[0008] Therefore, there is a continuing need to understand if there is a biochemical explanation for adaptation and, if so, if there may be ways to apply such understandings to identify compounds that may be more susceptible or less susceptible to olfactory adaptation in a manner that is quick and efficient. SUMMARY

[0009] The present disclosure relates to the discovery that the degree of cellular internalization of olfactory receptors following exposure to a modulator correlates reasonably well with the observation of olfactory adaptation in human sensory testing.

[0010] In a first aspect, the disclosure provides a method of identifying a substance that is susceptible to olfactory adaptation, the method comprising: (a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an agonist of the olfactory receptors; and (b) determining a degree of cellular internalization of the plurality of olfactory receptors. In some embodiments, the method further comprises: (c) identifying an adaptation-susceptible olfactory receptor agonist that is susceptible to olfactory adaptation based on the determined degree of cellular internalization. In some further embodiments, the method further comprises: (d) deselecting the adaptation-susceptible olfactory receptor agonist as an adaptation-resistant substance.

[0011] In a second aspect, the disclosure provides a method of identifying a substance that is susceptible to olfactory adaptation, the method comprising: (a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an antagonist of the olfactory receptors; and (b) determining a degree of cellular internalization of the plurality of olfactory receptors. In some embodiments, the introducing step comprises introducing the olfactory receptor ligand in the presence of a second olfactory ligand, wherein the second olfactory ligand is an agonist of the olfactory receptor. In some embodiments, the method further comprises: (c) identifying an adaptation-susceptible olfactory receptor antagonist that is susceptible to olfactory adaptation based on the determined degree of cellular internalization. In some further embodiments, the method further comprises: (d) deselecting the adaptation- susceptible olfactory receptor antagonist as an adaptation-resistant substance.

[0012] In a third aspect, the disclosure provides a method of identifying a substance that is resistant to olfactory adaptation, the method comprising: (a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an agonist of the olfactory receptors; and (b) determining a degree of cellular internalization of the plurality of olfactory receptors. In some embodiments, the method further comprises: (c) identifying an adaptation-resistant olfactory receptor agonist that is resistant to olfactory adaptation based on the determined degree of cellular internalization. In some further embodiments, the method further comprises: (d) selecting the adaptation-resistant olfactory receptor agonist as an adaptation-resistant substance. In a fourth aspect, the disclosure provides a method of identifying a substance that is resistant to olfactory adaptation, the method comprising: (a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an antagonist of the olfactory receptors; and (b) determining a degree of cellular internalization of the plurality of olfactory receptors. In some embodiments, the introducing step comprises introducing the olfactory receptor ligand in the presence of a second olfactory ligand, wherein the second olfactory ligand is an agonist of the olfactory receptor. In some embodiments, the method further comprises: (c) identifying an adaptation-resistant olfactory receptor antagonist that is resistant to olfactory adaptation based on the determined degree of cellular internalization. In some further embodiments, the method further comprises: (d) selecting the adaptation-resistant olfactory receptor antagonist as an adaptation-resistant substance.

[0013] In a fifth aspect, the disclosure provides the use of an olfactory-resistant substance identified or selected by the method of the previous aspects to provide a long-lasting fragrance to a fragranced article. In some embodiments, the fragranced article is a fiber, such as a natural or synthetic fiber. In some such embodiments, the fiber is comprised by a textile article, such as an article of clothing, a towel, bedding, upholstery, and the like. In some embodiments, the fragranced article is a personal care product, such as a household cleaning product, a commercial cleaning product, a dish detergent, a laundry detergent, a fabric softener, a scent booster, a shower gel, a shampoo, a hair conditioner, a hair-styling product, a skin care product, a cosmetic product, a deodorant, an antiperspirant, or a self-tanning product. In some embodiments, the fragranced article is a perfuming composition, such as a fine fragrance composition or a fragrance composition for use in various air-care products, such as plug-in diffusers, candles, perfuming reeds, room sprays, and the like.

[0014] Further aspects, and embodiments thereof, are set forth below in the Detailed Description, the Drawings, the Abstract, and the Claims.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following drawings are provided for purposes of illustrating various embodiments of the compositions and methods disclosed herein. The drawings are provided for illustrative purposes only and are not intended to describe any preferred compositions or preferred methods, or to serve as a source of any limitations on the scope of the claimed inventions.

[0017] FIG. 1 shows a schematic representation of the canonical signaling cascade initiated by G protein-coupled receptor (GPCR) activation. Upon ligand binding, the olfactory receptor activates the Golf protein, which in turn stimulates adenylyl cyclase (AC) to produce cyclic AMP (cAMP).The increase in cAMP leads to the opening of cAMP-gated sodium (Na+) and calcium (Ca2+) channels. Additionally, cAMP activates protein kinase A (PKA), which phosphorylates the transcription factor CREB. Phosphorylated CREB (pCREB) serves as a surrogate marker of GPCR activity.

[0018] FIG. 2 shows the results of an assessment of olfactory receptor activity by measuring cAMP activation and CREB phosphorylation. FIG. 2A dose-response curve showing the activation of the olfactory receptor OR7A17 by two odorant compounds, Anthamber Premium and Wolfwood. The Y-axis represents the cAMP HTRF ratio, and the X-axis shows the logarithmic concentration of the compounds. A decrease in signal indicates greater activation. Wolfwood demonstrates greater potency as an agonist of OR7A17 compared to Anthamber Premium. FIG. 2B shows a corresponding dose-response curve measuring CREB phosphorylation (pCREB intensity) in response to the same compounds. At equimolar concentrations, Wolfwood induces a higher level of CREB phosphorylation than Anthamber Premium, consistent with its greater potency observed in the cAMP assay.

[0019] FIG. 3 shows OR7A17 internalization. FIG. 3A shows the quantification of OR7A17 receptor activation in response to 8 odorant ligands. OR7A17 is a human olfactory receptor that is activated primarily by woody odorants. A heterologous cell line expressing OR7A17 and Golf protein, was treated with individual ligands and cAMP activity was measured using a commercially available plate reader-based assay. The graph shows potency of each compound on the X-axis, and efficacy (as measured by cAMP activity) on the Y-axis. cAMP activity level does not predict levels of adaptation. The numbers indicate compounds as specified in Table 1. FIG. 2B shows the quantification of OR7A17 receptor internalization in response to 8 odorant ligands. A heterologous cell line expressing OR7A17, Golf protein, and P-arrestin 2 was treated with individual ligands, fixed at one hour post-stimulation, and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta (spots) per cell. The bar graph shows the relative degree of internalization (spot formation) for each ligand, indicating differential receptor trafficking responses. The compound numbers on the X-axis correspond to those shown in FIG. 3A. FIG. 30 shows the sensory adaptation measurements for the same set of odorant ligands. Human subjects rated the perceived intensity of each odorant before and after a 3-minute continuous exposure on an olfactometer. Adaptation was calculated as the change in perceived intensity (pre-exposure minus post-exposure). The bar graph displays the magnitude of adaptation for each ligand, demonstrating that ligands inducing greater receptor internalization tend to produce greater perceptual adaptation. Testing was performed in pairs of odorants, as indicated by the bar patterns. The compound numbers on the X-axis correspond to those shown in FIG. 3A.

[0020] FIG. 4 shows OR10K1 internalization. FIG. 4A shows the quantification of OR10K1 receptor internalization in response to 3 odorant ligands. OR10K1 is a human olfactory receptor that is activated primarily by aldehydic odorants. A heterologous cell line expressing OR10K1 , Golf protein, and p-arrestin 2 was treated with individual ligands, fixed at one hour poststimulation, and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta per cell. The bar graph shows the relative degree of internalization (spot formation) for each ligand, indicating differential receptor trafficking responses. The numbers on the X-axis indicate compounds as specified in Table 1. FIG. 4B shows the sensory adaptation measurements for the same set of odorant ligands. Human subjects rated the perceived intensity of each odorant before and after a 3-minute continuous exposure on an olfactometer. Adaptation was calculated as the change in perceived intensity (pre-exposure minus post-exposure). The bar graph displays the magnitude of adaptation for each ligand, demonstrating that ligands inducing greater receptor internalization tend to produce greater perceptual adaptation. Testing was performed in pairs of odorants, as indicated by the bar patterns. The compound numbers on the X-axis correspond to those shown in FIG. 4B.

[0021] FIG. 5 shows OR10AB1 internalization. FIG. 5A shows the quantification of OR10AB1 receptor internalization in response to 2 odorant ligands. OR10AB1 is a human olfactory receptor that is activated primarily by floral odorants. A heterologous cell line expressing OR10AB1 , Golf protein, and p-arrestin 2 was treated with individual ligands, fixed at one hour post-stimulation, and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta per cell. The bar graph shows the relative degree of internalization (spot formation) for each ligand, indicating differential receptor trafficking responses. The numbers on the X-axis indicate compounds as specified in Table 1. FIG. 5B shows the sensory adaptation measurements for the same set of odorant ligands. Human subjects rated the perceived intensity of each odorant before and after a 3-minute continuous exposure on an olfactometer. Adaptation was calculated as the change in perceived intensity (pre-exposure minus post-exposure). The bar graph displays the magnitude of adaptation for each ligand, demonstrating that ligands inducing greater receptor internalization tend to produce greater perceptual adaptation. The compound numbers on the X-axis correspond to those shown in FIG. 5A.

[0022] FIG. 6 shows OR5AN1 internalization. FIG. 6A shows the quantification of OR5AN1 receptor internalization in response to various odorant ligands. OR5AN1 is a human olfactory receptor that is activated primarily by musky odorants. A heterologous cell line expressing OR5AN1 , Golf protein, and p-arrestin 2 was treated with individual ligands, fixed at one hour post-stimulation, and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta per cell. The bar graph shows the relative degree of internalization (spot formation) for each ligand, indicating differential receptor trafficking responses. The numbers on the X-axis indicate compounds as specified in Table 1. FIG. 6B shows how human subjects rated the perceived intensity of each odorant before and after a 3-minute continuous exposure on an olfactometer. Adaptation was calculated as the change in perceived intensity (pre-exposure minus post-exposure). The bar graph displays the magnitude of adaptation for each ligand, demonstrating that ligands inducing greater receptor internalization tend to produce greater perceptual adaptation. The compound numbers on the X-axis correspond to those shown in FIG. 6A. FIG. 60 shows an additional set of OR5AN1 agonists tested using the same methods as in (A) and ranked from highest to lowest internalization. FIG. 6D shows the human sensory testing of compounds shown in FIG. 60 using an olfactometer from a different vendor. Adaptation is calculated as a percentage decrease of detected odorant intensity compared to initial rating. The compounds are ranked from highest to lowest adapting, and the trend corresponds to FIG. 60.

[0023] FIG. 7 shows the addition of receptor antagonists enhances agonist-mediated internalization of OR7A17. FIG. 7A shows the quantification of OR7A17 receptor internalization in response to Agonist (Cmpd 26) in the presence of antagonists (Antag. 1 - Cmpd 25, Antag. 2 - Cmpd 27). The cells expressing the receptor were treated with Agonist at 2 concentrations (low and high) alone and in the presence of antagonists, fixed at 1 hour poststimulation, and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta per cell. The bar graph shows the relative degree of internalization (spot formation) for each ligand, indicating enhancement of OR7A17 internalization by the antagonists. FIG. 7B shows the sensory self- and cross-adaptation measurements for Agonist (Cmpd 26). Human subjects rated the perceived intensity of Agonist before and after a 5-minute continuous exposure to Agonist for self-adaptation, or one of two of antagonists (Antag. 1 - Cmpd 25, Antag. 2 - Cmpd 27) for cross-adaptation. Both antagonists are known not to activate OR7A17. The bar graph displays the magnitude of adaptation to Agonist, demonstrating that despite absence of activation of OR7A17, both antagonists are able to enhance perceptual adaptation to the Agonist. The white bar shows an inactive compound that does not activate or antagonize OR7A17, which has no effect on sensory adaptation to the Agonist.

[0024] FIG. 8 shows the addition of a receptor antagonist enhances agonist-mediated internalization of 0R11A1 (FIG. 8A), OR5AN1 (FIG. 8B), and OR10J5 (FIG. 8C). A heterologous cell line expressing each individual receptor, Golf protein, and p-arrestin 2 was treated with the indicated ligands, fixed at one hour post-stimulation, and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta per cell. The bar graphs show the relative degree of internalization (spot formation) for each ligand, indicating differential receptor trafficking responses. White bars indicate negative control treatment, black bars indicate addition of agonist alone and gray bars indicate simultaneous addition of agonist and antagonist of the given receptor. Similar to the example given in FIG. 6, addition of antagonist increased receptor internalization for all 3 receptors tested, suggesting that signaling modulation can affect levels of adaptation to an odorant.

[0025] FIG. 9 shows internalization of multiple receptors activated by a mixture of compounds can predict sensory adaptation to mixtures. FIG. 9A shows the quantification of receptor internalization in response to two 5-ingredient accords (mixtures) and negative control (DMSO). A heterologous cell line expressing each individual receptor, Golf protein, and p-arrestin 2 was treated with one of two accords or negative control, fixed at one hour post-stimulation, and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta per cell. The heatmap shows the relative degree of internalization (spot formation) of each receptor in response to each accord. Dark shades indicate low internalization, and light shades indicate high internalization for each receptor. FIG. 9B shows the sensory adaptation measurements for the 2 accords. Human subjects rated the perceived intensity of each accord before and after a 3-minute continuous exposure on an olfactometer. Adaptation was calculated as the change in perceived intensity (pre-exposure minus postexposure). The bar graph displays the magnitude of adaptation for each mixture, demonstrating that mixtures inducing greater receptor internalization (across multiple receptors) tend to produce greater perceptual adaptation.

[0026] DETAILED DESCRIPTION

[0027] The following Detailed Description sets forth various aspects and embodiments provided herein. The description is to be read from the perspective of the person of ordinary skill in the relevant art. Therefore, information that is well known to such ordinarily skilled artisans is not necessarily included.

[0028] Definitions

[0029] The following terms and phrases have the meanings indicated below, unless otherwise provided herein. This disclosure may employ other terms and phrases not expressly defined herein. Such other terms and phrases have the meanings that they would possess within the context of this disclosure to those of ordinary skill in the art. In some instances, a term or phrase may be defined in the singular or plural. In such instances, it is understood that any term in the singular may include its plural counterpart and vice versa, unless expressly indicated to the contrary. As used herein, the term “polypeptide” refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain of two or more amino acids and does not refer to a specific length of the chain. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used herein to refer to a chain of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with, any of these terms.

[0030] As used herein, the term “polynucleotide” refers to a compound that includes a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (namely, cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (II)), a sugar (namely, deoxyribose or ribose), and a phosphate group. In general, the polynucleotide is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the polynucleotide. The sequence of bases is typically represented from 5’ to 3’. Herein, the term polynucleotide encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular, messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers including two or more of these molecules. The polynucleotide may be linear or circular. In addition, the term polynucleotide includes both sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the polynucleotides described herein can contain naturally occurring or non-naturally occurring nucleotides.

[0031] As used herein, the term “functional fragment” refers to a portion of a polypeptide sequence to which the ligand binds. Polypeptide sequences often contain certain amino acids that do not actively participate in binding, but which may serve other purposes. In some instances, these non-functioning parts of the polypeptide sequence can be removed or partially replaced, while leaving the functional portion of the sequence intact. These modified proteins are said to comprise a functional fragment of the original polypeptide sequence.

[0032] As used herein, the term “adaptation” refers to a reduction in perceived intensity to a compound or a substance after exposure to a compound or a substance for prolonged periods of time such as minutes, hours or days.

[0033] As used herein, the term “ligand” refers to a compound or substance that binds orthosterically or allosterically as an agonist or an antagonist to an olfactory receptor, when the olfactory receptor expressed in a eukaryotic cell in a cell-based assay.

[0034] As used herein, the term “olfactory receptor agonist” refers to a compound or substance that binds as an agonist to an olfactory receptor, when the olfactory receptor expressed in a eukaryotic cell in a cell-based assay, with an EC50 of no more than 600 pM. As used herein, the term “olfactory receptor antagonist” refers to a compound or substance that binds as an antagonist to an olfactory receptor, when the olfactory receptor expressed in a eukaryotic cell in a cell-based assay, with an IC50 of no more than 600 pM.

[0035] As used herein, the term “allele” refers to one of several alternative forms of a gene or polynucleotide sequence at a specific chromosomal location (locus).

[0036] As used herein, the term “human OR7A17 olfactory receptor” or “OR7A17 receptor” refers to the polypeptide sequence that in humans is encoded by the OR7A17 alleles, whose sequence is well known in the art.

[0037] As used herein, the term “human OR5AN1 olfactory receptor” or“OR5AN1 receptor” refers to the polypeptide sequence that in humans is encoded by the OR5AN1 alleles, whose sequence is well known in the art.

[0038] As used herein, the term “human OR10G4 olfactory receptor” or “OR10G4 receptor” refers to the polypeptide sequence that in humans is encoded by the OR10G4 alleles, whose sequence is well known in the art.

[0039] As used herein, the term “human OR10K1 olfactory receptor” or “OR10K1 receptor” refers to the polypeptide sequence that in humans is encoded by the OR10K1 alleles, whose sequence is well known in the art.

[0040] As used herein, the term “human OR10AB1 olfactory receptor” or“OR10AB1 receptor” refers to the polypeptide sequence that in humans is encoded by the OR10AB1 alleles, whose sequence is well known in the art.

[0041] As used herein, the term “human OR11 A1 olfactory receptor” or “OR11A1 receptor” refers to the polypeptide sequence that in humans is encoded by the OR11A1 alleles, whose sequence is well known in the art.

[0042] As used herein, the term “human OR10J5 olfactory receptor” or “OR10J5 receptor” refers to the polypeptide sequence that in humans is encoded by the OR10J5 alleles, whose sequence is well known in the art.

[0043] As used herein, the term “Anthamber Premium” refers to the compound having the name ((+-)-1-(2,3,8,8-tetramethyl-1 ,2,3,4,5,6,7,8-octahydro-2-naphthalenyl)ethenone.

[0044] As used herein, the term “Wolfwood” refers to the compound having the name ((+)-(1S,2S,3S,5R)-2,6,6-trimethylspiro[bicyclo[3.1.1]heptane-3,T-cyclohexane]-2'-en-4'-one)

[0045] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a substituent” encompasses a single substituent as well as two or more substituents, and the like.

[0046] As used herein, “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. Unless otherwise expressly indicated, such examples are provided only as an aid for understanding embodiments illustrated in the present disclosure and are not meant to be limiting in any fashion. Nor do these phrases indicate any kind of preference for the disclosed embodiment.

[0047] As used herein, “comprise” or “comprises” or “comprising” or “comprised of” refer to groups that are open, meaning that the group can include additional members in addition to those expressly recited. For example, the phrase, “comprises A” means that A must be present, but that other members can be present too. The terms “include,” “have,” and “composed of” and their grammatical variants have the same meaning. In contrast, “consist of” or “consists of” or “consisting of” refer to groups that are closed. For example, the phrase “consists of A” means that A and only A is present.

[0048] As used herein, “optionally” means that the subsequently described event(s) may or may not occur. In some embodiments, the optional event does not occur. In some other embodiments, the optional event does occur one or more times.

[0049] As used herein, “or” is to be given its broadest reasonable interpretation and is not to be limited to an either / or construction. Thus, the phrase “comprising A or B” means that A can be present and not B, or that B is present and not A, or that A and B are both present. Further, if A, for example, defines a class that can have multiple members, e.g., Ai and A2, then one or more members of the class can be present concurrently.

[0050] Other terms are defined in other portions of this description, even though not included in this subsection.

[0051] Screening Methods

[0052] In certain aspects, the disclosure provides a method of identifying a substance that is susceptible to olfactory adaptation, the method comprising: (a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an agonist of the olfactory receptors; and (b) determining a degree of cellular internalization of the plurality of olfactory receptors. In some embodiments, the method further comprises: (c) identifying an adaptation-susceptible olfactory receptor agonist that is susceptible to olfactory adaptation based on the determined degree of cellular olfactory receptor internalization. In some further embodiments, the method further comprises: (d) deselecting the adaptation-susceptible olfactory receptor agonist as an adaptation-resistant substance.

[0053] In another aspect, the disclosure provides a method of identifying a substance that is susceptible to olfactory adaptation, the method comprising: (a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an antagonist of the olfactory receptors; and (b) determining a degree of cellular internalization of the plurality of olfactory receptors. In some embodiments, the introducing step comprises introducing the olfactory receptor ligand in the presence of a second olfactory ligand, wherein the second olfactory ligand is an agonist of the olfactory receptor. In some embodiments, the method further comprises: (c) identifying an adaptation-susceptible olfactory receptor antagonist that is susceptible to olfactory adaptation based on the determined degree of cellular internalization. In some further embodiments, the method further comprises: (d) deselecting the adaptation- susceptible olfactory receptor antagonist as an adaptation-resistant substance.

[0054] In certain related aspects, the disclosure provides a method of identifying a substance that is resistant to olfactory adaptation, the method comprising: (a) introducing an olfactory receptor agonist to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor agonist is an agonist of the olfactory receptors; and (b) determining a degree of cellular internalization of the plurality of olfactory receptors. In some embodiments, the method further comprises: (c) identifying an adaptation-resistant olfactory receptor agonist that is resistant to olfactory adaptation based on the determined degree of cellular internalization. In some further embodiments, the method further comprises: (d) selecting the adaptation-resistant olfactory receptor agonist as an adaptation-resistant substance.

[0055] In yet another aspect, the disclosure provides a method of identifying a substance that is resistant to olfactory adaptation, the method comprising: (a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an antagonist of the olfactory receptors; and (b) determining a degree of cellular internalization of the plurality of olfactory receptors. In some embodiments, the introducing step comprises introducing the olfactory receptor ligand in the presence of a second olfactory ligand, wherein the second olfactory ligand is an agonist of the olfactory receptor. In some embodiments, the method further comprises: (c) identifying an adaptation-resistant olfactory receptor antagonist that is resistant to olfactory adaptation based on the determined degree of cellular internalization. In some further embodiments, the method further comprises: (d) selecting the adaptation-resistant olfactory receptor antagonist as an adaptation-resistant substance.

[0056] The foregoing screening methods are not limited to any particular olfactory receptor or its ligands. It is believed that the methods are generalizable to a wide range of olfactory receptors in addition to those for which working Examples are provided. For example, in some embodiments, the plurality of olfactory receptors are a plurality of human olfactory receptors, such as a plurality of human OR7A17, 0R5AN1, OR10K1, OR10AB1, 0R11A1 , OR10J5 olfactory receptors, or a polypeptide whose amino acid sequence is at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 99%, equivalent thereto.

[0057] Any suitable olfactory receptor ligand can be used in the screening method, so long as the compound or substance is a ligand of at least one of the plurality of olfactory receptors employed in the method. For example, in embodiments where the plurality of olfactory receptors are a plurality of human OR7A17 olfactory receptors, the olfactory receptor ligand is a human OR7A17 olfactory receptor ligand. In general, the particular ligands of various human olfactory receptors are well known in the art. Moreover, such ligands can be identified without undue experimentation by a person of skill in the art using standard cellular screening assays, where the olfactory receptor is expressed in a eukaryotic cell and test compounds or substances are introduced to the assay and an activation or inhibition dose-response curve is generated. The skilled artisan can readily calculate an EC50 from the resulting activation curve or an IC50 for the inhibition curve. In general, compounds or substances having an activation curve with respect to an olfactory receptor with an EC50 of no more than 600 pM are classified as agonists of the olfactory receptor in question. In general, compounds or substances having an inhibition curve with respect to an olfactory receptor with an IC50 of no more than 600 pM are classified as antagonists of the olfactory receptor in question.

[0058] Examples of suitable olfactory receptor ligands for use in the methods described herein include, not are not limited to ((+-)-1-(2,3,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydro-2- naphthalenyl)ethanone and ((+)-(1 S,2S,3S,5R)-2,6,6-trimethylspiro[bicyclo[3.1.1 ]heptane-3, 1 cyclohexane]-2'-en-4'-one). In general, the plurality of olfactory receptors is expressed on the surface of a plurality of cells. In general, the eukaryotic cells are engineered to overexpress the polypeptides corresponding to the olfactory receptor agonist, a functional fragment thereof, or a polypeptide having at least 70% equivalence to the foregoing. The eukaryotic cells can be engineered to overexpress any of the plurality of olfactory receptors, such as the human OR7A17 olfactory receptor. In some embodiments, the eukaryotic cells are engineered to overexpress one or more of the polypeptides corresponding to different olfactory receptors.

[0059] In some embodiments, the eukaryotic cell further comprises an exogenous nucleic acid sequence that encodes for a G protein. In some further embodiments, the G-protein is the a- subunit Golf or any chimera thereof. In some further embodiments, the G protein is gustducin, such as an a-gustducin. In some other embodiments, the G protein is a promiscuous G protein. Non-limiting examples of promiscuous G proteins include Ga16, Ga15, Ga16gust25, Ga15gust25, Ga16gust44, Ga15gust44, and Ga15-i / 3-5, which are described in detail in U.S. Patent No. 10,107,794. In some other embodiments, the G protein is a G(i / o) protein.

[0060] In some embodiments, the eukaryotic cell further comprises a G protein coupled to the olfactory receptors. In some further embodiments, the G-protein is the a-subunit Golf or any chimera thereof. In some further embodiments, the G protein is gustducin, such as an a- gustducin. In some other embodiments, the G protein is a promiscuous G protein. Non-limiting examples of promiscuous G proteins include Ga16, Ga15, Ga16gust25, Ga15gust25, Ga16gust44, Ga15gust44, and Ga15-i / 3-5, which are described in U.S. Patent No. 10,107,794. In some other embodiments, the G protein is a G(i / o) protein. Any suitable cell can be used. For example, human H EK-293 and human LI2OS cells are well known in the art and are generally available commercially. In some embodiments, the eukaryotic cells are isolated eukaryotic cells. In general, the eukaryotic cells are contained in an assay. In some embodiments, the eukaryotic cells are adhered to a substrate. Various functional assays can be used, including assays that measure of changes in intracellular calcium cation concentration (for example, Fluorometric Imaging Plate Reader-based Ca2+mobilization assay, FLIPR), intracellular cyclic adenosine monophosphate (cAMP) (for example Homogenous Time Resolved Fluorescence, Pherastar), pERK1 / 2 activation (for example, high content imaging, HCI), and receptor internalization (for example, TRANSFLUOR). A change in olfactory receptor activity by a test compound or by an olfactory receptor ligand indicates modulation of the olfactory receptor by the test compound or the agonist.

[0061] In some instances, cells besides human HEK-293 or human U2OS cells can also be used. Non-limiting examples of such cells include the following cell types: 1A2, ARH-77, RWPE-1, WI-38, EJM, NCI-H1155, L-1236, NCI-H526, JM1, SHP-77, SNU-878, NCI-H2196, C3A, CA46, SNU-466, KS-1, SNU-738, MOLP-2, HDLM-2, Pfeiffer, HCC-15, Alexander cells, L-540, KMS-12-BM, JK-1, NCI-H1092, SW 1990, NCI-H1184, SU-DHL-1, Hep 3B2.1-7, P3HR-1, NCI-H2029, SU-DHL-5, SNU-1, MOLP-8, SUP-M2, MONO-MAC-1 , SNU-1040, KYM- 1 , HEC-59, HCC1569, OCI-LY3, Hs 819.T, DU4475, CI-1 , S-117, OVCAR-8, SNU-626, HL-60, SUIT-2, T3M-4, RKO, MOR / CPR, DK-MG, GA-10, OCUM-1, HCT-15, HT, MONO-MAC-6, G- 402, Toledo, COV362, SU-DHL-8, Daoy, NCI-H1435, LS513, Hs 839.T, Hs 172.T, BT-483, KMS-21 BM, AGS, NCI-H2172, LC-1 / sq-SF, SNU-201 , NUGC-4, SK-HEP-1 , SUP-B15, SNU-5, HT-1197, SUP-T1, AMO-1 , KU812, AN3 CA, AML-193, VMRC-RCW, HLE, HuH28, Hs 751.T, NCI-H2110, MEG-01 , MV-4-11, Hep G2, KYSE-30, KALS-1, BICR 6, RMUG-S, JHH-6, Ki-JK, IST-MES1 , HCC-95, HPB-ALL, HSC-3, 697, LOU-NH91, KARPAS-299, GI-1, COLO 792, SK-N-FI, D341 Med, HGC-27, SR-786, COLO-818, MHH-CALL-2, SF126, NCI-H322, A-253, NCI-H1623, MCF7, HCC-44, FU97, OCI-LY-19, Hs 766T, NCI-H522, RL, HCC1428, RPMI 6666, U-937, NCI-H460, SW 1088, NCI-H1792, NCI-H1693, UACC-257, JHUEM-2, HuT 78, UACC-893, NCI-H929, A-704, OV56, LN-229, OE19, SK-MEL-24, RD-ES, NCI-H211, KCI- MOH1, NCI-H1963, Hs 706.T, ChaGo-K-1, EPLC-272H, OPM-2, KHM-1 B, A549, HuG1-N, NCI-H508, MHH-CALL-3, SNU-1076, A3 / KAW, MEL-HO, TO 175.T, Caki-1 , Hs 936.T, SK-LU-1, WM-983B, K-562, EFE-184, SNU-520, NCI-H2291 , HCC-1195, ABC-1, KE-39, NH-6, HCC2218, CMK, RS4;11, KYSE-450, OV7, KYSE-510, SK-UT-1, SNU-C1 , OE33, P12- ICHIKAWA, DLD-1, COV434, HuNS1, SNU-899, SW480, COLO-678, LU99, KOPN-8, NCI-H2227, SW1463, Hs 675.T, JHH-4, NCI-H1703, HEC-1-A, BDCM, MIA PaCa-2, PC-3, TE-15, PK-45H, MKN-45, HCC-366, CAL-29, HEC-50B, CPC-N, KMRC-20, SW1116, EOL-1 , COLO 205, EHEB, YD-38, MC116, SK-N-BE(2), BV-173, NCI-H2347, LU65, RT4, U-87 MG, LK-2, KP-N-YN, HEC-251 , NCI-H1651 , GP2d, RERF-LC-MS, NB-4, NCI-H2286, SNU-61 , T- 47D, huH-1, KYSE-180, ST486, SW 1353, M-07e, KASUMI-1, YH-13, NCI-H28, GAMG, JeKo-1, GOS-3, SNU-324, PA-TU-8902, MFE-280, SNU-245, NALM-1, RERF-LC-Sq1, BICR22, ZR-75-1, COR-L23, SW579, COR-L88, KM12, Hs611.T, OUMS-23, RERF-LC-Ad1, NCI-H1385, SK-LMS-1, COLO-320, BL-70, GRANTA-519, MCAS, Pane 08.13, AM-38, KMS-11, SIG-M5, SNU-407, JHOS-2, OVCAR-4, Set-2, OV-90, MeWo, HEL, HT-29, MDA-MB-231, T0V-21G, NCI-H1355, KMS-27, NALM-6, KMS-26, Caov-4, KASUMI-2, UACC-62, U266B1, Hs 695T, HT55, BICR 31, TCC-PAN2, KMS-20, Hs 578T, RI-1, Hs 606.T, NCI-H1341, THP-1, BCP-1, Hs 737.T, SW1417, MOLT-4, Raji, ESS-1, MEL-JUSO, SH-10-TC, Hs 683, ME-1, EB2, PLC / PRF / 5, NCI-H1339, A4 / Fuk, SEM, HEC-265, IST-MES2, KE-97, NCI-H1437, COLO-704, NCI-H1915, TE-5, NCI-H2023, NCI-H82, T1-73, SNU-840, HuT 102, NCI-H1944, KYSE-520, Kasumi-6, 1321N1, Hs 742.T, IM95, PL45, CL-40, WM1799, KMM-1, SNU-449, JHUEM-1, KARPAS-620, Loucy, SNU-1079, Daudi, HCC-56, HSC-2, COR-L47, PA-TU-89885, OAW28, COR-L311, L-363, Malme-3M, NOMO-1, Hs 870.T, SU-DHL-10, Hs 229.T, NCI-H810, KYSE-410, RPMI-8402, SNU-175, EBC-1, RVH-421, K029AX, PA-TU-8988T, LXF-289, OVSAHO, CAL-12T, Hs 940.T, MM1-S, SUP-HD1, LNCaP clone FGC, HSC-4, NU-DHL-1, NCI-H2228, BEN, CAL-78, Sq-1, NCI-H1793, SNU-C2A, M DA- MB- 134- VI, COV318, KE-37, TYK-nu, MOTN-1, T98G, SW837, EB1, Becker, PE / CA-PJ34 (clone C12), Hs 616.T, NCI-H446, WM-88, CHP-126, Calu-1, SNU-283, NCI-H1573, SW 1271, SNU-16, JHOS-4, ACHN, Calu-3, KMRC-1, SW 1783, TE-11, TE-9, HuH-6, P31 / FUJ, HT-1376, NCI-H520, 786-0, KNS-60, Caki-2, OVK18, PL-21, NCI-H2452, JURL-MK1, TEN, JHH-7, MDA-MB-157, Calu-6, RKN, NUGC-2, ONS-76, J82, OUMS-27, SNU-1196, Hs 739.T, RPMI-7951, NCI-H854, JHH-5, JVM-2, Hey-A8, 5637, KYSE-140, Capan-2, KYSE-150, HEC-1-B, BICR 16, HEL 92.1.7, MHH-NB-11, SNU-387, SK-OV-3, SK-MEL-28, IGROV1, ML-1, HLF-a, CHL-1, YKG1, A-204, OCI-M1, 8505C, JVM-3, NCI-H647, DB, COLO-800, PK-59, FaDu, HLF, OVMANA, EFO-27, PF-382, NCI-H747, LS123, SU-DHL-6, SJRH30, PANC-1, NCI-H2342, KM-H2, DND-41, HH, HuCCTI, F-36P, DMS 454, Hs 274.T, AU565, NCI-H1666, EN, RH-41, NCI-H1373, NCI-H838, SK-MEL-30, MOLM-6, DEL, NCI-H226, NCI-H1648, NCI-H661, 143B, Mino, C32, KMS-34, NCI-H1694, SK-ES-1, UACC-812, GDM-1, NCI-H23, Pane 02.03, CCF-STTG1, LOXIMVI, SJSA-1, MDST8, PK-1, NCI-H716, SU-DHL-4, MPP 89, MJ, COLO 829, PE / CA-PJ15, HD-MY-Z, BxPC-3, WM-793, COLO 668, T84, JHOM-1, PEER, LS411N, GMS-10, KMBC-2, RMG-I, KELLY, SNU-761, NALM-19, HEC-151, G-361, OVTOKO, A-498, SW900, LCLC-103H, FTC-133, QGP-1, Reh, CMK-11-5, NU-DUL-1, BT-20, Hs 600.T, Hs 604.T, KATO III, SNU-410, NCI-H2126, SK-MEL-5, MDA-MB-468, AsPC-1, HUP-T3, KP-N-SI9s, L-428, SNU-1105, HUP-T4, 769-P, LMSU, NCI-H1869, NCO2, MOLM-16, CAL 27, HCC70, NCI-H1930, COV644, Hs 863.T, HCC-2279, D283 Med, Hs 944.T, HCC1599, MDA-MB-415, HCC2157, NCI-H1618, SNU-308, HCC1954, DMS 153, HPAF-II, T24, CJM, VM-CUB1, UM-UC-3, LAMA-84, NCI-H1734, JHH-2, VMRC-RCZ, MFE-319, MDA-MB-453, SNU-503, T0V-112D, B-CPAP, GSU, HCC-78, NCI-H2171 , CAMA-1 , HEC-108, HCC4006, CAL-85-1, NCI-H2122, COLO-699, NCI-H196, LUDLU-1, SW 780, RPMI 8226, LP-1, PC-14, HuTu 80, T.T, SW948, 22Rv1, HARA, NCI-H596, IPC-298, SCaBER, NCI-H1838, NB-1, Hs 934.T, Hs 895.T, DMS 114, KYSE-70, KP-3, KP4, DAN-G, NCI-H2009, OC 316, SCC-25, U-138 MG, RCC10RGB, MFE-296, NCI-H1755, RERF-LC-KJ, 8305C, WSU-DLCL2, ES-2, MSTO-211 H, SCC-15, ZR-75-30, PSN1 , SNU-423, NCI-H2106, TE-1 , UT-7, KMS-28BM, NCI-H2081 , SK-MM-2, COLO 741, OC 314, HCC1395, MOLT-13, LN-18, Pane 10.05, PE / CA-PJ41 (clone D2), Hs 746T, CW-2, SKM-1, NUGC-3, TE-10, NCI-H358, NCI-H69, BFTC-909, HOS, BICR 18, NCI-H1395, OVKATE, Hs 698.T, EFM-19, COLO-783, MHH-CALL-4, ACC-MESO-1, NCI-H1436, KP-N-RT-BM-1, SK-MEL-31 , NCI-H1105, CAL-51 , YD-15, NCI-H2085, NCI-H2444, HCC1187, Hs 939.T, CAL-120, SCC-9, TUHR14TKB, KMRC-2, KG-1-C, ECC10, CGTH-W-1, NCI-H841 , C2BBe1 , SUP-T11, RCH-ACV, CADO-ES1, JURKAT, 647-V, SK-MEL-2, MDA-MB-175-VII, MKN74, SNU-C4, LCLC-97TM1 , SCC-4, BHY, IGR-37, KYO-1, Hs 281.T, TT, TUHR4TKB, HT-1080, NCI-H660, TE 441.T, LS1034, KNS-42, Pane 04.03, HCC1419, AZ-521, SNG-M, NCI-N87, G-292, clone A141B1, KPL-1, MDA-MB-361 , CL-14, NCI-H2170, HuH-7, RD, NCI-H2066, IGR-1, TE-14, VCaP, BL-41, SNU-620, SK-MES-1, MEC-2, NCI-H1299, IGR-39, RT112 / 84, SF-295, DV-90, A2780, BICR 56, NCI-H510, NCI-H2141 , YD-8, NCI-H2405, TF-1, MEC-1, CCK-81 , NCI- H1048, Hs 822.T, NCI-H2052, K052, CAL-54, Hs 840.T, SW620, SK-CO-1, BT-474, CL-11, KNS-62, NCI-H1650, G-401, MOLT-16, SNU-398, COLO-680N, EM-2, Hs 294T, CAL-62, KMRC-3, A101 D, KG-1, BT-549, HT115, A-375, SW-1710, WM-115, KLE, JHUEM-3, MKN7, CHP-212, HCC202, BC-3C, NCI-H1568, KMS-18, PE / CA-PJ49, COLO-849, SIMA, OCI-AML3, GSS, EC-GI-10, EFO-21, RCM-1 , DMS 273, KU-19-19, RERF-GC-1 B, SH-4, SK-MEL-3, RERF-LC-Ad2, M059K, JHOM-2B, MDA PCa 2b, Hs 852.T, RL95-2, Pane 03.27, SNU-216, Pane 02.13, CFPAC-1, SK-N-SH, OCI-AML2, LoVo, SBC-5, NCI-H1876, NCI-H441, SK-N-AS, COR-L24, HCC38, NCI-H1781, DOHH-2, NCI-H1563, U-251 MG, HPAC, JIMT-1, U-2 OS, A- 673, TC-71, NCI-H650, NIH:OVCAR-3, CAS-1, JL-1, SK-MEL-1 , MDA-MB-4355, Ishikawa (Heraklio) 02 ER-, TE 617.T, SU.86.86, RERF-LC-AI, TT2609-C02, LS 180, YAPC, HDQ-P1, KNS-81 , FU-OV-1, KP-2, DMS 53, SNU-1272, Detroit 562, 42-MG-BA, L3.3, COLO-679, NCI- H2087, NCI-H2030, GCT, NCI-H889, Caov-3, MDA-MB-436, NCI-H524, MKN1, KCL-22, Capan-1, CML-T1, H4, NCI-H727, Hs 343.T, MHH-ES-1, NMC-G1 , HCC-1171, REC-1, Hs 618.T, A172, YD-10B, SW48, MUTZ-5, TE-6, JHH-1 , HCT 116, TE-4, IA-LM, MG-63, NCI- H1975, TALL-1, HCC1806, HMCB, SCLC-21H, HCC1500, CL-34, Pane 05.04, SW403, TM-31 , HCC1937, JMSU-1 , DMS 79, SNB-19, NCI-H1836, Li-7, HCC827, 639-V, MOLM-13, SK-BR-3, IMR-32, TUHR10TKB, OAW42, SK-N-MC, TGBC11TKB, NCI-H1581, EFM-192A, YMB-1 , HCC2935, ECC12, HCC-33, DU 145, NCI-H146, SNU-1214, SNU-1077, 23132 / 87, HT-144, SNU-182, Hs 888.T, SNU-475, GCIY, Hs 729, JHOC-5, SW 1573, HEC-6, OCI-AMLS, Hs 688(A). T, HS 821.T, PCM6, RT-112, SK-N-DZ, SNU-478, SNU-119, HCC1143, NCI-H209, 8- MG-BA, COR-L105, COR-L95, SNU-46, COV504, CAL-148, SNU-05, DBTRG-05MG, BHT- 101 , WM-266-4, BFTC-905, KYSE-270, TE-8, SNU-213, and SH-SYSY.

[0062] In many cases, it may take some amount of time for the olfactory receptors expressed on the surface of the cell to be internalized by the cell following the initial introduction of the olfactory receptor ligand to the olfactory receptor. Thus, in some embodiments, the introducing step (a) includes waiting for a period of time to allow time for internalization to occur. For example, in some embodiments, the waiting time ranges from as short as 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, or 30 minutes to as long as 90 minutes, 120 minutes, or 180 minutes. In some embodiments, the waiting time ranges from 30 minutes to 120 minutes.

[0063] The methods set forth herein include a step of determining a degree of cellular internalization of the plurality of olfactory receptors. Suitable means of assessing cellular internalization include, for example, genetically tagging the olfactory receptor with a fluorescent tag (such as the red fluorescent protein mCherry2) or performing immunostaining with chemical fluorescent probes for the olfactory receptor to assess its subcellular localization. For example, in some embodiments, phospho-CREB (pCREB) intensity may be measured in the nuclei of mCherry2 positive cells. In some embodiments, the determining step comprises imaging of the cells comprising an olfactory receptor and assessing changes to the cells that are indicative of internalization of the olfactory receptor. Such assessed changes include: 1) measuring intensity of the fluorescently labeled olfactory receptor in different subcellular regions (i.e. whole cell, cytoplasm, cell membrane, intracellular vesicles, nuclei); 2) measuring a number of high content imaging parameters such as the “texture” of fluorescence signal, where a rougher texture would indicate olfactory receptor internalization; 3) Measuring number / size / fluorescence intensity of subcellular spots that indicate uptake of olfactory receptor through the endocytic pathway. Using multiple fluorescence features, a population of responding cells can be identified and calculated as a percentage of the total cell population. In some embodiments, the plurality of olfactory receptors are modified with a tag, such as a fluorescent tag. Examples of fluorescent tags suitable for use in tagging olfactory receptors include, but are not limited to GFP, YFP, RFP, CFP, DsRed2, mCherry2, tdTomato, and mRuby3. In some embodiments, the determining comprises obtaining fluorescent images of the cells comprising the olfactory receptors before introduction of the olfactory receptor ligand and at a period of time following introduction. For example, in embodiments where the olfactory receptor is modified with a fluorescent tag, spot intensity of the fluorescent tag can be measured. A higher value for the measured spot intensity indicates greater internalization, while a lower value for the measured spot intensity indicates lower internalization. For any particular olfactory receptor and a series of olfactory receptor ligands, the spot intensities of the various agonists can be compared at comparable concentrations and wait times to determine which ligands tend to promote cellular internalization of the olfactory receptor and which do not.

[0064] In some further embodiments of any of the foregoing embodiments, the methods further comprise identifying an adaptation-susceptible olfactory receptor ligand that is susceptible to olfactory adaptation based on the determined degree of cellular olfactory receptor internalization or an adaptation-resistant olfactory receptor ligand that is resistant to olfactory adaptation, based on the determined degree of cellular internalization from step (b). The ligands shown to promote internalization of the olfactory receptor are classified as adaptation- susceptible agonists, and those ligands shown not to promote significant internalization of the olfactory receptor are classified as adaptation-resistant agonists. The skilled artisan is capable of making such distinctions based on differences in the determined degree of internalization for a series of ligands of a particular olfactory receptor.

[0065] In some further embodiments, the methods further comprise selecting adaptationresistant ligands or deselecting adaptation-susceptible ligands for use in various applications where olfactory adaptation may be undesirable.

[0066] The olfactory receptor agonists can be any suitable compound or substance that is amenable for use in cellular screening assays. For example, in some embodiments, the olfactory receptor ligand is an organic compound. In some embodiments, the olfactory receptor ligand is a naturally occurring compound. In some other embodiments, the olfactory receptor ligand is a non-naturally occurring compound. In some embodiments, the olfactory receptor ligand is a peptide, such as a polypeptide or an oligopeptide, an amino acid, an amino acid derivative, an amide, a nucleotide, an oligonucleotide. In some embodiments, the olfactory receptor ligand is a plant or food extract, such as a plant or food extract obtained by bioassay- guided fractionation. In some embodiments, the olfactory receptor ligand is a terpene, a diterpene or a triterpene. In some embodiments, the olfactory receptor ligands is a ribonucleotide or derivative thereof. In some embodiments, the olfactory receptor ligand is an organic molecule having a molecular weight of no more than 1000 amu.

[0067] Uses and Methods

[0068] In other aspects, the disclosure provides use of an olfactory-resistant substance identified or selected by the method of the foregoing aspects to provide a long-lasting fragrance to a fragranced article. The disclosure also provides methods that correspond to each of the foregoing uses. Thus, in certain related aspects, the disclosure provides methods of providing a long-lasting fragrance to a fragranced article, the method comprising introducing to an article an adaptation-resistant substance identified or selected by the method of the foregoing aspects. In certain related aspects, the disclosure provides methods of providing a long-lasting fragrance to a fragranced article, the method comprising removing from an article an adaptation-susceptible substance identified or deselected by the method of the foregoing aspects. Any suitable fragranced articles are contemplated. In some embodiments, the fragranced article is a fiber, such as a natural or synthetic fiber. In some such embodiments, the fiber is comprised by a textile article, such as an article of clothing, a towel, bedding, upholstery, and the like. In some embodiments, the fragranced article is a personal care product, such as a household cleaning product, a commercial cleaning product, a dish detergent, a laundry detergent, a fabric softener, a scent booster, a shower gel, a shampoo, a hair conditioner, a hair-styling product, a skin care product, a cosmetic product, a deodorant, an antiperspirant, or a self-tanning product. In some embodiments, the fragranced article is a perfuming composition, such as a fine fragrance composition or a fragrance composition for use in various air-care products, such as plug-in diffusers, candles, perfuming reeds, room sprays, and the like.

[0069] Fiber and Textiles

[0070] In certain related aspects, the disclosure provides uses to impart a long-lasting fragrance to a fiber, the method comprising introducing one or more selected or identified substances to the fiber. In general, the fiber is a textile fiber, such as a natural or synthetic fiber comprised by a textile article, such as an article of clothing, a towel, bedding, upholstery, and the like. In some embodiments, the identified or selected substance is introduced to the fiber as part of a perfuming composition included within a personal care composition, such as those described in further detail below.

[0071] Consumer Care Compositions and Related Uses and Products

[0072] In certain related aspects, the disclosure provides uses to impart a long-lasting fragrance to a consumer care composition, the method comprising introducing one or more selected or identified substances to the consumer care composition.

[0073] Further embodiments of the consumer care compositions referenced in connection with the foregoing methods and uses are set forth in more detail below.

[0074] In certain aspects, the disclosure provides a consumer care composition comprising a plurality of microparticles of the first aspect or any embodiments thereof. In some embodiments, the consumer care composition is in the form of a household cleaning product, a commercial cleaning product, a dish detergent, a laundry detergent, a fabric softener, a scent booster, a shower gel, a shampoo, a hair conditioner, a hair-styling product, a skin care product, a cosmetic product, a deodorant, an antiperspirant, or a self-tanning product.

[0075] In some embodiments, the consumer care composition comprises a plurality of microparticles and at least one active ingredient, such as an active ingredient selected from the group consisting of a cosmetic ingredient, skin caring ingredient, perfume ingredient, flavor ingredient, malodor counteracting ingredient, bactericide ingredient, fungicide ingredient, pharmaceutical or agrochemical ingredient, a sanitizing ingredient, an insect repellent or attractant, and mixtures thereof.

[0076] In some embodiments, the consumer care composition is a perfuming composition. In some such embodiments, the consumer care composition comprises a plurality of microparticles, as defined above where the microparticle is a coacervate core-shell microcapsule whose core comprises at least one perfume compound, and, optionally, at least one perfumery adjuvant or liquid perfumery carrier.

[0077] Liquid perfumery carriers are well known in the art. Some non-limiting examples include an emulsifying system, such as a solvent and a surfactant system, or a solvent commonly used in perfumery. Some non-limiting examples of suitable such solvents include dipropyleneglycol, diethyl phthalate, isopropyl myristate, benzyl benzoate, 2-(2-ethoxyethoxy)-1 -ethanol or ethyl citrate, which are the most commonly used. For the compositions which comprise both a perfumery carrier and a perfumery co-ingredient, other suitable perfumery carriers than those previously specified, can be also ethanol, water / ethanol mixtures, limonene or other terpenes, isoparaffins such as those known under the trademark ISOPAR (Exxon Chemical, Houston, Tex., US) or glycol ethers and glycol ether esters such as those known under the trademark DOWANOL (Dow Chemical Company, Midland, Mich., US). By “perfumery co-ingredient” it is meant here a compound, which is used in a perfuming preparation or a composition to impart a hedonic effect and which is not a microcapsule as defined above. In other words such a co- ingredient, to be considered as being a perfuming one, must be recognized by a person skilled in the art as being able to at least impart or modify in a positive or pleasant way the odor of a composition, and not just as having an odor.

[0078] Perfuming co-ingredients are well known in the art. In general terms, these perfuming co-ingredients belong to chemical classes as varied as alcohols, lactones, aldehydes, ketones, esters, ethers, acetates, nitriles, terpenoids, nitrogenous or sulfurous heterocyclic compounds and essential oils, and said perfuming co-ingredients can be of natural or synthetic origin. Many of these co-ingredients are in any case listed in reference texts such as the book by Arctander, PERFUME AND FLAVOR CHEMICALS (1969) or its more recent versions, or in other works of a similar nature, as well as in the abundant patent literature in the field of perfumery. It is also understood that said co-ingredients may also be compounds known to release in a controlled manner various types of perfuming compounds. Non-limiting examples of co-ingredients include 4-(dodecylthio)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-butanone, 4-(dodecylthio)-4- (2,6,6-trimethyl-1-cyclohexen-1-yl)-2-butanone, trans-3-(dodecylthio)-1-(2,6,6-trimethyl-3- cyclohexen-1-yl)-1-butanone, 2-(dodecylthio)octan-4-one, 2-phenylethyl oxo(phenyl)acetate, 3,7-dimethylocta-2,6-dien-1-yl oxo(phenyl)acetate, (Z)-hex-3-en-1-yl oxo(phenyl)acetate, 3,7- dimethyl-2,6-octadien-1-yl hexadecanoate, bis(3,7-dimethylocta-2,6-dien-1-yl) succinate, (2-((2- methylundec-1-en-1-yl)oxy)ethyl)-benzene, 1-methoxy-4-(3-methyl-4-phenethoxybut-3-en-1- yl)benzene, (3-methyl-4-phenethoxybut-3-en-1-yl)benzene, 1-(((Z)-hex-3-en-1-yl)oxy)-2- methylundec-1-ene, (2-((2-methylundec-1-en-1-yl)oxy)ethoxy)benzene, 2-methyl-1-(octan-3- yloxy)undec-1 -ene, 1 -methoxy-4-(1 -phenethoxyprop-1 -en-2-yl)benzene, 1 -methyl-4-(1 - phenethoxyprop- 1-en-2-yl)benzene, 2-(1-phenethoxyprop-1-en-2-yl)naphthalene, (2- phenethoxyvinyl)benzene, 2-(1-((3,7-dimethyloct-6-en-1-yl)oxy)prop-1-en-2-yl)naphthalene, (2- ((2-pentylcyclopentylidene)-methoxy)ethyl)-benzene, 4-allyl-2-methoxy-1-((2-methoxy-2- phenylvinyl)oxy)benzene, (2-((2-heptyl-cyclopentylidene)methoxy)ethyl)benzene, 1-isopropyl-4- methyl-2-((2-pentyl-cyclopentylidene)methoxy)benzene, 2-methoxy-1-((2- pentylcyclopentylidene)methoxy)-4-propylbenzene, 3-methoxy-4-((2-methoxy-2- phenylvinyl)oxy)benzaldehyde, 4-((2-(hexyloxy)-2-phenylvinyl)oxy)-3-methoxybenzaldehyde or a mixture thereof or a mixture thereof.

[0079] The term “perfumery adjuvant” refers to an ingredient capable of imparting additional added benefit such as a color, a particular light resistance, chemical stability, etc. Such compounds are well known in the art.

[0080] The personal care composition can contain any suitable amount of the microparticles of the present disclosure. In some embodiments, the personal care composition comprises from 0.01% by weight to 30% by weight of microparticles, based on the total weight of the consumer care composition.

[0081] The consumer care composition can be in any suitable physical state, such as a solid (such as a powder), liquid, or a gas. In some embodiments, the consumer care composition is a liquid. In some such embodiments, the liquid consumer care composition has one or more of the following characteristics: a) comprises from 2% by weight to 65% by weight of a surfactant, based on the total weight of the consumer care composition; b) comprises water or a water-miscible hydrophilic organic solvent; c) is in the form of a microparticle slurry; and d) comprises a non-encapsulated perfume.

[0082] In some embodiments, the consumer care composition is in the form of a powder. In some such embodiments, the powder consumer care composition has one or more of the following characteristics: a) comprises from 2% by weight to 65% by weight of a surfactant, based on the total weight of the consumer care composition; b) is in the form of a microcapsule powder; and c) comprises a perfume powder that is different from any perfume comprised by the microparticles.

[0083] In embodiments where the plurality of microparticles comprise encapsulated cores containing perfume compounds, the consumer care composition comprising these microparticles can be of used in various perfumed consumer products, such as products belonging to fine fragrance or “functional” perfumery. Functional perfumery includes personal care products, including hair care products, body cleansing products, skin care products, hygiene products, as well as home care products, including laundry care products, surface care products, and air care products. The term “perfumed consumer product” broadly refers to any consumer product that is expected to deliver, among different benefits, a perfuming effect to the surface to which it is applied, including, but not limited to, skin, hair, textiles, paper, countertops, sinks, toilets, floors, furniture, or other household surfaces, or in the air, for example, in the case of an air freshener, a room deodorizer, a candle, a reed diffuser, and the like.

[0084] Such perfumed consumer products can contain any other ingredients that are commonly used in the industry. Methods of formulating perfume-containing microcapsules are such products is also well known and can be used to develop formulations of such products containing microparticles of the present disclosure.

[0085] Non-limiting examples of suitable perfumed consumer products include a perfume, such as a fine perfume, a cologne, an after-shave lotion, a body splash, a fabric care product, such as a liquid or solid detergent, tablets and unit dose (single or multi-chambers), a fabric softener, a dryer sheet, a fabric refresher, an ironing water, a bleach, a hair care product, such as a shampoo, a hair conditioner, a coloring preparation, or a hair spray, a cosmetic preparation, such as a vanishing cream, a body lotion, or a deodorant or antiperspirant, or a skin-care product, such as a perfumed soap, a shower or bath mousse, a body wash, an oil or gel, bath salts, or a hygiene product, an air care product, such as an air freshener or a “ready to use” powdered air freshener, or a home care product, such all-purpose cleaners, liquid or power or tablet dishwashing products, toilet cleaners, or products for cleaning various surfaces, for example sprays and wipes intended for the treatment or refreshment of textiles or hard surfaces like floors, tiles, and stone, or a hygiene product such as sanitary napkins, diapers, or toilet paper.

[0086] In some embodiments, the consumer care composition comprises: a personal care active base, and a plurality of microparticles according to any of the embodiments set forth herein, wherein the consumer care composition is in the form of a personal care product.

[0087] Any suitable personal care active base or combination of such materials can be used. Such materials are well known to the skilled artisan and are widely discussed in the relevant patent literature. Non-limiting examples of a consumer care active base include surfactants, oils, hydrophobic solvents, hydrophilic solvents, water, and the like, as well as auxiliary agents, such as bleaching agents, buffering agent, builders, soil release or soil suspension polymers, granulated enzyme particles, corrosion inhibitors, antifoaming, sud suppressing agents, dyes, fillers, and mixtures thereof. In some embodiments, the personal care product is a hair-care product, such as a shampoo, a hair conditioner, a coloring preparation, or a hair spray, a cosmetic preparation such as a vanishing cream, a body lotion, or a deodorant or antiperspirant, or a skin care product, such as a perfumed soap, a shower, or a bath mousse, a body wash, an oil or gel, bath salts, or a hygiene product.

[0088] In some embodiments, the consumer care composition comprises: a home care active base, and a plurality of microparticles according to any of the embodiments set forth herein, wherein the consumer care composition is in the form of a home care product.

[0089] Any suitable home care active base or combination of such materials can be used. Such materials are well known to the skilled artisan and are widely discussed in the relevant patent literature. Non-limiting examples of a consumer care active base include surfactants, oils, hydrophobic solvents, hydrophilic solvents, water, and the like, as well as auxiliary agents, such as bleaching agents, buffering agent, builders, soil release or soil suspension polymers, granulated enzyme particles, corrosion inhibitors, antifoaming, sud suppressing agents, dyes, fillers, and mixtures thereof.

[0090] In some embodiments, the home care product is an air care product, such as an air freshener or a “ready to use” powdered air freshener, or a home care product, such all-purpose cleaners, liquid or power or tablet dishwashing products, toilet cleaners, or products for cleaning various surfaces, for example sprays and wipes intended for the treatment or refreshment of textiles or hard surfaces like floors, tiles, and stone, or a hygiene product such as sanitary napkins, diapers, or toilet paper.

[0091] In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1% by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition.

[0092] The consumer care composition can have any suitable pH. For example, in some embodiments, the consumer care composition has a pH of less than 7. In some other embodiments, the consumer care product has a pH of at least 7.

[0093] Fabric Softener

[0094] In some embodiments, the consumer care composition is in the form of a fabric softener composition. In some such embodiments, the consumer care composition comprises a fabric softener active base and a plurality of microparticles according to any of the embodiments set forth above.

[0095] Any suitable fabric softener active base can be used. For example, in some embodiments, the fabric softener active base includes dialkyl quaternary ammonium salts, dialkyl ester quaternary ammonium salts, 1,2-dioleoyl-3-trimethylammonium propane, triethanolamine quaternary salts, silicones, and mixtures thereof, as well as various surfactants, hydrophilic organic solvents, and water. In some embodiments, the consumer care composition comprises the fabric softener active base in an amount ranging from 85% by weight and 99.95% by weight, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1 % by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises nonencapsulated perfume compounds.

[0096] Liquid Detergent

[0097] In some embodiments, the consumer care composition is in the form of a liquid detergent composition. In some such embodiments, the consumer care composition comprises a liquid detergent active base and a plurality of microparticles according to any of the embodiments set forth above.

[0098] Any suitable liquid detergent active base can be used. For example, in some embodiments, the liquid detergent active base includes anionic surfactant such as alkylbenzenesulfonate (ABS), secondary alkyl sulfonate (SAS), primary alcohol sulfate (PAS), lauryl ether sulfate (LES), methyl ester sulfonate (MES) and nonionic surfactant such as alkyl amines, alkanolamide, fatty alcohol poly(ethylene glycol) ether, fatty alcohol ethoxylate (FAE), ethylene oxide (EO) and propylene oxide (PO) copolymers, amine oxydes, alkyl polyglucosides, alkyl polyglucosamides, as well as various surfactants, hydrophilic organic solvents, and water. In some embodiments, the consumer care composition comprises the liquid detergent active base in an amount ranging from 85% by weight and 99.95% by weight, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1% by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises non-encapsulated perfume compounds.

[0099] Solid Detergent

[0100] In some embodiments, the consumer care composition is in the form of a solid detergent composition. In some such embodiments, the consumer care composition comprises a solid detergent active base and a plurality of microparticles according to any of the embodiments set forth above.

[0101] Any suitable solid detergent active base can be used. For example, in some embodiments, the solid detergent active base includes anionic surfactant such as alkylbenzenesulfonate (ABS), secondary alkyl sulfonate (SAS), primary alcohol sulfate (PAS), lauryl ether sulfate (LES), methyl ester sulfonate (MES) and nonionic surfactant such as alkyl amines, alkanolamide, fatty alcohol poly(ethylene glycol) ether, fatty alcohol ethoxylate (FAE), ethylene oxide (EO) and propylene oxide (PO) copolymers, amine oxydes, alkyl polyglucosides, alkyl polyglucosamides, as well as various surfactants, hydrophilic organic solvents, water, and fatty acid carboxylates. In some embodiments, the consumer care composition comprises the solid detergent active base in an amount ranging from 85% by weight and 99.95% by weight, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1 % by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises non-encapsulated perfume compounds.

[0102] Shampoo or Shower Gel

[0103] In some embodiments, the consumer care composition is in the form of a shampoo or shower gel composition. In some such embodiments, the consumer care composition comprises a shampoo or shower gel active base and a plurality of microparticles according to any of the embodiments set forth above.

[0104] Any suitable shampoo or shower gel active base can be used. For example, in some embodiments, the shampoo or shower gel active base includes anionic surfactant such as sodium alkylether sulfate, ammonium alkylether sulfates, alkylamphoacetate, cocamidopropyl betaine, cocamide MEA, alkylglucosides and aminoacid based surfactants and mixtures thereof, as well as various surfactants, hydrophilic organic solvents, and water. In some embodiments, the consumer care composition comprises the shampoo or shower gel active base in an amount ranging from 85% by weight and 99.95% by weight, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1% by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises non-encapsulated perfume compounds.

[0105] Rinse-Off Conditioner

[0106] In some embodiments, the consumer care composition is in the form of a rinse-off conditioner composition. In some such embodiments, the consumer care composition comprises a rinse-off conditioner active base and a plurality of microparticles according to any of the embodiments set forth above.

[0107] Any suitable rinse-off conditioner active base can be used. For example, in some embodiments, the rinse-off conditioner active base includes cetyltrimonium chloride, stearyl trimonium chloride, benzalkonium chloride, behentrimonium chloride and mixture thereof, as well as various surfactants, hydrophilic organic solvents, and water. In some embodiments, the consumer care composition comprises the rinse-off conditioner active base in an amount ranging from 85% by weight and 99.95% by weight, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1% by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises non-encapsulated perfume compounds.

[0108] Solid Scent Booster

[0109] In some embodiments, the consumer care composition is in the form of a solid scent booster composition. In some such embodiments, the consumer care composition comprises a solid scent booster active base and a plurality of microparticles according to any of the embodiments set forth above.

[0110] Any suitable solid scent booster active base can be used. For example, in some embodiments, the solid scent booster active base includes urea, sodium chloride, sodium sulphate, sodium acetate, zeolite, sodium carbonate, sodium bicarbonate, clay, talc, calcium carbonate, magnesium sulfate, gypsum, calcium sulfate, magnesium oxide, zinc oxide, titanium dioxide, calcium chloride, potassium chloride, magnesium chloride, zinc chloride, saccharides such as sucrose, mono-, di-, and polysaccharides and derivatives such as starch, cellulose, methyl cellulose, ethyl cellulose, propyl cellulose, polyols / sugar alcohols such as sorbitol, maltitol, xylitol, erythritol, and isomalt, PEG, PVP, citric acid or any water soluble solid acid, fatty alcohols or fatty acids and mixtures thereof, as well as various surfactants, hydrophilic organic solvents, and water. In some embodiments, the consumer care composition comprises the solid scent booster active base in an amount ranging from 85% by weight and 99.95% by weight, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1 % by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises non-encapsulated perfume compounds.

[0111] Liquid Scent Booster

[0112] In some embodiments, the consumer care composition is in the form of a liquid scent booster composition. In some such embodiments, the consumer care composition comprises a liquid scent booster active base and a plurality of microparticles according to any of the embodiments set forth above. Any suitable liquid scent booster active base can be used. For example, in some embodiments, the liquid scent booster active base includes ethoxylated aliphatic alcohols, POE / PPG (polyoxyethylene and polyoxypropylene) ethers, mono and polyglyceryl esters, sucrose ester compounds, polyoxyethylene hydroxylesters, alkyl polyglucosides, amine oxides, alcohols, salts and esters of carboxylic acids, salts and esters of hydroxyl carboxylic acids, fatty acids, fatty acid salts, glycerol fatty acids, and mixtures thereof, as well as various surfactants, hydrophilic organic solvents, and water. In some embodiments, the consumer care composition comprises the liquid scent booster active base in an amount ranging from 85% by weight and 99.95% by weight, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1% by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises nonencapsulated perfume compounds.

[0113] Hair Colorant

[0114] In some embodiments, the consumer care composition is in the form of a hair colorant composition. In some such embodiments, the consumer care composition comprises a hair colorant active base and a plurality of microparticles according to any of the embodiments set forth above.

[0115] Any suitable hair colorant active base can be used. For example, in some embodiments, the hair colorant active base includes oxidizing agents, an alkakine agent, dye precursors, coupling agents, as well as various surfactants, hydrophilic organic solvents, and water. In some embodiments, the consumer care composition comprises the hair colorant active base in an amount ranging from 85% by weight and 99.95% by weight, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1% by weight to 15 % by weight, or from 0.2% by weight to 5% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the consumer care composition comprises non-encapsulated perfume compounds.

[0116] Perfuming Composition

[0117] In some embodiments, the consumer care composition is in the form of a perfuming composition. In some such embodiments, the consumer care composition comprises perfume compounds, ethanol, and a plurality of microparticles according to any of the embodiments set forth above. In some embodiments, the consumer care composition comprises the plurality of microparticles in an amount ranging from 0.1% by weight to 30 % by weight, or from 0.2% by weight to 20% by weight, of the plurality of microparticles, based on the total weight of the consumer care composition. In some embodiments, the perfume compounds are present in an amount ranging from 0% by weight to 40% by weight, or from 3% by weight to 40% by weight, based on the total weight of the consumer care composition. In some embodiments, the ethanol is present in an amount ranging from 20% by weight to 90% by weight, or from 40% by weight to 90% by weight, based on the total weight of the consumer care composition.

[0118] EXAMPLES

[0119] To further illustrate this invention, the following examples are included. The examples should not, of course, be construed as specifically limiting the invention. Variations of these examples within the scope of the claims are within the purview of one skilled in the art and are considered to fall within the scope of the invention as described and claimed herein. The reader will recognize that the skilled artisan, armed with the present disclosure, and skill in the art is able to prepare and use the invention without exhaustive examples.

[0120] Methodology

[0121] Cell-based olfactory receptor activation assay

[0122] A cell-based homogeneous time-resolved fluorescence (HTRF) assay was used to quantify intracellular cyclic adenosine monophosphate (cAMP) levels as an indicator of olfactory receptor activation in a human cell line. Cells were plated in 96-well opaque white plates and transiently transfected with a plasmid encoding the olfactory receptor of interest, and a separate plasmid encoding Golf protein (an olfactory-specific G-protein involved in downstream signal transduction). At 24 hours after transfection, the cells were treated with previously characterized agonists for 30 minutes in the presence of 3-isobutyl-1 -methylxanthine (IBMX) to prevent cAMP degradation. Following treatment, cAMP activation was measured using a commercially available plate reader-based HTRF cAMP detection kit (revvity).

[0123] Cell-based olfactory receptor internalization assay

[0124] To evaluate the adaptation of olfactory receptors (ORs) to various odorant compounds, a cell-based assay was developed using a human cell line. The cells were seeded into 96-well clear bottom plates and transiently transfected with a plasmid encoding the olfactory receptor of interest, along with separate plasmids to express the Golf protein, and p-arrestin 2 (ARRB2), which plays a role in receptor internalization and desensitization.

[0125] FIG. 1 shows a schematic representation of the canonical signaling cascade initiated by GPCR activation. Upon ligand binding, the olfactory receptor activates the Golf protein, which in turn stimulates adenylyl cyclase (AC) to produce cyclic AMP (cAMP). The increase in cAMP leads to the opening of cAMP-gated sodium (Na+) and calcium (Ca2+) channels. Additionally, cAMP activates protein kinase A (PKA), which phosphorylates the transcription factor CREB. Phosphorylated CREB (pCREB) serves as a surrogate marker of GPCR activity. Following transfection, the cells were incubated under standard cell culture conditions. On the subsequent day, each well was treated with a distinct odorant compound, allowing for high throughput screening of multiple ligands. The cells were exposed to the odorants at 37 °C for one hour to permit receptor-ligand interaction and potential receptor internalization.

[0126] After incubation, the cells were fixed using paraformaldehyde to preserve cellular structures. Standard immunocytochemistry techniques were employed to label the olfactory receptors, enabling visualization of their subcellular localization. In addition, the cells were immunostained for pCREB as a downstream activity marker to identify the population of cells responding to the odorants. Imaging was performed using a high-throughput fluorescence microscope (Cytation C10), which allowed for rapid acquisition of high-resolution images across multiple wells. FIG. 2B shows a dose-response curve measuring CREB phosphorylation (pCREB intensity) in response to Anthamber Premium and Wolfwood. At equimolar concentrations, Wolfwood induces a higher level of CREB phosphorylation than Anthamber Premium, consistent with its greater potency observed in the cAMP assay (FIG. 2A).

[0127] Immunostained receptor internalization was assessed via Cytation Gen5 analysis software by quantifying the number of intracellular OR-positive spots per cell, which served as a proxy for endosomal uptake. An increase in intracellular receptor signal was interpreted as evidence of ligand-induced internalization, suggesting that the corresponding odorant may promote olfactory adaptation.

[0128] Human sensory panel testing of adaptation to odorants

[0129] To measure the degree of adaptation for a given odorant in vivo, static and dynamic olfactometry methods were used. The static olfactometry method entailed depositing 250 pl of diluted odorant onto a cellulose pad in a glass jar. Each jar was capped for four hours to achieve an equilibrated headspace. Dynamic olfactometry involved making Nalophan bags filled with nitrogen and 1 pl of volatilized odorant; the bag was then attached to an air-dilution Scentroid olfactometer which was used to deliver the odorant simultaneously to six sensory panelists at the desired concentration.

[0130] The panelists were instructed to rate the scent intensity in a jar on a scale of 1-10 by taking 1-3 sniffs of the jar headspace. After a 1-minute timeout, each panelist positioned their nose directly above an olfactometer port, and the olfactometer delivered the stream of the same odorant as in the jar for a 3-minute adaptation period. T o minimize variations in breathing frequency, for the entire period, the panelists inhaled and exhaled through the nose in synchrony with a recurring musical cue that was played continuously on a computer. As a check on whether the desired intensity was achieved, the panelists rated the intensity of the odorant stream in the beginning of the adaptation period.

[0131] Immediately after the adaptation period ended, the panelists rated scent intensity in two additional jars, one of which contained the same odorant, whereas the other was “a blank” (i.e. , contained no odorant but was visually identical to a jar with the odorant). The order in which the blank versus the odorant jar were evaluated varied from panel to panel. This was done to minimize the panelists’ expectation that pre- versus post-adaptation evaluations always involved the same odorant. For a given odorant, the degree of adaptation was measured by subtracting its mean post-adaptation intensity from its pre-adaptation mean. For each odorant, the means were based on evaluations of at least 20 panelists. Dilution ratios used in the jars and olfactometers were chosen based on pilot evaluations and as intended, resulted in odorant concentrations that were roughly iso-intense before adaptation.

[0132] Example 1 - OR7A17 Activation

[0133] Eukaryotic cells were plated in white opaque plates and transfected with a human OR7A17 olfactory receptor and Golf protein. On the following day, the cells were treated with two prospective OR7A17 agonists for 30 minutes. The two agonists used were Anthamber Premium (Firmenich SA, Satigny, Switzerland) and Wolfwood (Firmenich SA, Satigny, Switzerland). Activation of cAMP was measured using a commercially available plate readerbased assay (revvity). The assay was performed in the presence of IBMX to prevent cAMP degradation. FIG. 2A shows the cAMP HTRF ratio as a function of agonist concentration, where a decrease in cAMP HTRF ratio indicates increasing concentration of generated cAMP. Treatment with Wolfwood resulted in a lower EC50 value and a larger HTRF Ratio span, indicating that it is a more potent and efficacious agonist compared to Anthamber Premium.

[0134] To assess further downstream signaling, eukaryotic cells were plated in clear-bottom black imaging plates and transfected with an mCherry2-tagged version of the human OR7A17 olfactory receptor. The cells were treated with Anthamber Premium or Wolfwood for 60 minutes and fixed using standard paraformaldehyde fixation protocols. The cells were immunostained for pCREB. The plates were imaged on a high content imager (Cytation C10), and image analysis was performed using Gen5 software that is included with the Cytation C10 instrument. To determine the level of OR7A17 activation, pCREB intensity was measured in the nuclei of OR7A17-mCherry2 positive cells. FIG. 2B shows pCREB intensity as a function of agonist concentration. Consistent with data in the cAMP assay, Wolfwood treatment resulted in a lower EC50 and higher activity of CREB phosphorylation compared to Anthamber Premium, indicating greater potency and efficacy.

[0135] Example 2 - OR7A17 Internalization Assay

[0136] To evaluate the ability of the receptor internalization assay to predict olfactory adaptation, eight previously characterized agonists of human OR7A17 receptor were tested for receptor activation using the cAMP HTRF assay (FIG. 3A). As in Example 1 , eukaryotic cells were transfected with a human OR7A17 olfactory receptor and Golf protein, treated with individual ligands, and cAMP activity was measured using the HTRF plate reader-based assay. The graph shows potency of each compound on the X-axis and efficacy on the Y-axis. The numbers indicate compounds as specified in Table 1.

[0137] Table 1

[0138] Using the microscopy-based assay, receptor internalization was measured in response to the same 8 agonists that were tested in the cAMP HTRF assay. At 1-hour post stimulation with individual agonists, the cells were fixed and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta (spots) per cell in that subpopulation. The bar graph in FIG. 3B shows the relative degree of internalization (spot formation) for each ligand, indicating differential receptor trafficking responses. The compound numbers on the X-axis correspond to those shown in FIG. 3A. Based on comparison of OR7A17 activation and internalization data, cAMP activity level does not predict levels of receptor internalization.

[0139] To validate ability of receptor internalization assay to predict human sensory adaptation to OR agonists, human subjects rated the perceived intensity of each agonist before and after a 3-minute continuous exposure on an olfactometer. Adaptation was calculated as the change in perceived intensity (pre-exposure minus post-exposure). The bar graph inf FIG. 3C displays the magnitude of adaptation for each ligand, demonstrating that ligands inducing greater receptor internalization tend to produce greater perceptual adaptation. Testing was performed in pairs of agonists, as indicated by the bar patterns. The compound numbers on the X-axis correspond to those shown in FIG. 3A and FIG. 3B.

[0140] To demonstrate wide applicability of predicting sensory adaptation to OR agonists across different olfactory tonalities, three more receptors were tested. All assays were run using the same methods indicated for OR7A17, substituting only the transfected receptor. All numbers on the X-axes indicate compounds as specified in Table 1. FIG. 4 shows OR10K1 internalization and sensory adaptation to aldehydic compounds. FIG. 5 shows OR10AB1 internalization and sensory adaptation to floral compounds. FIG. 6 shows OR5AN1 internalization and sensory adaptation to musk compounds. In the case of OR5AN1, two independent human studies used slightly different sensory testing protocols. FIG. 6A and FIG. 6B show a pairwise comparison of two pairs of agonists, and FIG. 60 and FIG. 6D show a ranking of five agonists from most to least adapting. Compounds 14 and 15 rank as high in both graphs, whereas compounds 17 and 18 rank as low in both graphs. All tested receptors demonstrated that receptor internalization assay can accurately predict sensory adaptation to OR agonists.

[0141] Example 3 - Adaptation Prediction

[0142] To evaluate the ability of the receptor internalization assay to predict effects of OR antagonists on adaptation, OR7A17 receptor internalization was quantified in response to an agonist (Cmpd 26) in the absence and presence of antagonists (Antag. 1 is Cmpd 25, and Antag. 2 is Cmpd 27). The cells expressing the receptor were treated with an agonist at two concentrations (low and high) alone, and at a low concentration of agonist in the presence of antagonists. The cells were fixed at 1-hour post-stimulation, and immunostained for the receptor. High-content imaging was used to identify pCREB-positive cells, and receptor internalization was assessed by quantifying the number of receptor-positive endosomal puncta per cell. The bar graph in FIG. 7A shows the relative degree of internalization (spot formation) in response to each treatment, indicating enhancement of agonist-mediated internalization of OR7A17 by addition of antagonists. There is no statistically significant difference between the effects of the two antagonists.

[0143] Receptor internalization data was compared to sensory data of self- and crossadaptation to the agonist (Cmpd 26). Human subjects rated the perceived intensity of the agonist before and after a 5-minute continuous exposure to agonist for self-adaptation, or a 5-minute continuous exposure to one of two antagonists for cross-adaptation (Antag. 1 is Cmpd 25, Antag. 2 is Cmpd 27). Both antagonists are known not to activate OR7A17. The bar graph in FIG. 7B displays the magnitude of adaptation to agonist, demonstrating that despite absence of activation of OR7A17, both antagonists are able to enhance perceptual adaptation to the agonist. Consistent with the data from receptor internalization assay, there is no statistically significant difference between the effects of the two antagonists. The white bar shows an inactive compound that does not activate or antagonize OR7A17 and has no effect on sensory adaptation to the agonist.

[0144] Receptor internalization assay was repeated for 3 additional receptors: OR11 A1 , OR5AN1 and OR10J5 (FIG. 8). The following compounds were used in FIG. 8: (A) Agonist - Cmpd 22, Antagonist - Cmpd 18. (B) Agonist - Cmpd 14, Antagonist - Cmpd 18. (C) Agonist - Cmpd 23, Antagonist - Cmpd 24. Similar to the example given in FIG. 7, addition of an antagonist increased receptor internalization for all 3 receptors tested, suggesting that signaling modulation can affect levels of adaptation to an agonist. Example 4 - Sensory Testing

[0145] To determine whether receptor internalization assay can be used to predict sensory adaptation to mixtures of ingredients, two 5-ingredient accords were tested. Based on prior data about OR activation by these agonist ingredients, six individual ORs were identified for testing in the assay. Each mixture (accord) was applied individually to each OR and receptor internalization was measured and quantified. The heatmap in FIG. 9A shows the relative degree of internalization (spot formation) of each receptor in response to each accord. Dark shades indicate low internalization, and light shades indicate high internalization for each receptor. Accord 2 promotes internalization of more receptors and to a greater extent than Accord 1. Consistent with this, FIG. 9B shows sensory adaptation measurements for the 2 accords. The bar graph displays the magnitude of adaptation for each mixture, demonstrating that mixtures inducing greater receptor internalization (across multiple receptors) promote greater perceptual adaptation.

Claims

CLAIMS1. A method of identifying a substance that is susceptible to olfactory adaptation, the method comprising:(a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an agonist of the olfactory receptors; and(b) determining a degree of cellular internalization of the plurality of olfactory receptors.

2. The method of claim 1 , wherein the olfactory receptor ligand is an olfactory receptor agonist.

3. The method of claim 1 , wherein the olfactory receptor ligand is an olfactory receptor antagonist, and the introducing comprises introducing the olfactory receptor ligand to the plurality of olfactory receptors in the presence of an olfactory receptor agonist.

4. The method of any one of claims 1 to 3, further comprising: (c) identifying an adaptation- susceptible olfactory receptor ligand that is susceptible to olfactory adaptation based on the determined degree of cellular internalization.

5. The method of claim 4, further comprising: (d) deselecting the adaptation-susceptible olfactory receptor ligand as an adaptation-resistant substance.

6. A method of identifying a substance that is resistant to olfactory adaptation, the method comprising:(a) introducing an olfactory receptor ligand to a plurality of olfactory receptors, wherein each of the plurality of olfactory receptors is expressed on a surface of a cell, and wherein the olfactory receptor ligand is an agonist of the olfactory receptors; and(b) determining a degree of cellular internalization of the plurality of olfactory receptors.

7. The method of claim 6, wherein the olfactory receptor ligand is an olfactory receptor agonist.

8. The method of claim 6, wherein the olfactory receptor ligand is an olfactory receptor antagonist, and the introducing comprises introducing the olfactory receptor ligand to the plurality of olfactory receptors in the presence of an olfactory receptor agonist.

9. The method of any one of claims 6 to 8, further comprising: (c) identifying an adaptationresistant olfactory receptor ligand that is resistant to olfactory adaptation based on the determined degree of cellular internalization.

10. The method of claim 9, further comprising: (d) selecting the adaptation-resistant olfactory receptor ligand as an adaptation-resistant substance.

11. The method of any one of claims 1 to 10, wherein the olfactory receptor is selected from the group consisting of: the human OR7A17, OR5AN1, OR10K1 , OR10AB1 , OR11A1 , OR10J5 olfactory receptors, and any polypeptide having an amino acid sequence that is at least 70% equivalent thereto.

12. Use of an adaptation-resistant substance identified or selected by the method of claim 4, 5, 9, or 10 to provide a long-lasting fragrance to a fragranced article.

13. The use of claim 12, wherein the fragranced article is a fiber, such as a synthetic or natural fiber.

14. The use of claim 12, wherein the fragranced article is a consumer care product, such as a household cleaning product, an air freshener, a commercial cleaning product, a dish detergent, a laundry detergent, a fabric softener, a scent booster, a shower gel, a shampoo, a hair conditioner, a hair-styling product, a skin care product, a cosmetic product, a deodorant, an antiperspirant, or a self-tanning product.

15. The use of claim 12, wherein the fragranced article is a perfuming composition, such as a fine fragrance composition.

16. A method of providing a long-lasting fragrance to a fragranced article, the method comprising introducing an adaptation-resistant substance identified or selected by the method of claim 4, 5, 9, or 10 to the fragranced article.

17. A method of providing a long-lasting fragrance to a fragranced article, the method comprising removing an adaptation-susceptible substance identified or selected by the method of claim 2, 3, 7, or 8 to the fragranced article.

18. The method of claim 16 or 17, wherein the fragranced article is a fiber, such as a synthetic or natural fiber.

19. The method of claim 16 or 17, wherein the fragranced article is a consumer care product, such as a household cleaning product, an air freshener, a commercial cleaning product, a dish detergent, a laundry detergent, a fabric softener, a scent booster, a shower gel, a shampoo, a hair conditioner, a hair-styling product, a skin care product, a cosmetic product, a deodorant, an antiperspirant, or a self-tanning product.

20. The method of claim 16 or 17, wherein the fragranced article is a perfuming composition, such as a fine fragrance composition.

21. A perfuming composition for providing long-lasting fragrance, which comprises an adaptation-resistant substance identified or selected by the method of claim 2, 3, 5, or 6.

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