Methods for identifying positive allosteric modulators of odorant receptors

Abstract

The various aspects presented herein relate to the fragrance industry. More specifically, the various aspects presented herein relate to assays and methods based on the use of specific olfactory receptors activated by target odorant compounds to screen and identify compositions and / or ingredients that enhance a subject's perception of a target odorant compound.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial Nos. 62 / 609,004 and 62 / 609,017, filed on December 21, 2017, U.S. Provisional Patent Application Serial No. 62 / 781,796, filed on December 19, 2018, and European Patent Application Serial No. 18157690.1, filed on February 20, 2018, and European Patent Application Serial No. 18166887.2, filed on April 11, 2018, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The technical field relates to odorants and aroma receptors, and assays useful for identifying odorants and / or aroma compounds, more particularly positive modulators of human odorant receptor activity. Background Art

[0004] The sense of smell is the most complex and poorly understood of the human sensory systems. There are many steps from olfactory receptor (OR) activation to perception that still require further investigation. If we can understand how the OR code for individual odorants and mixtures translates into perception, we can use this knowledge to bring significant benefits in multiple areas. These areas include: odor modulators, such as malodor antagonists, which block unpleasant odors; new flavoring (food flavors) and fragrance (daily fragrance) ingredients, which replace non-biodegradable or toxic compounds; and odor enhancers, which will limit our dependence on source compounds that are difficult to obtain from natural sources or will improve sensitivity to certain fragrance ingredients. Central to the combinatorial paradigm of the "olfactory receptor code" is the observation that any single OR can be activated by multiple odorants, and conversely, most odorants are able to activate several ORs. In the mouse genome, there are about 1,200 different complete ORs. In contrast, humans have about 400. In both cases, all components of the OR are activated by the thousands of odorants in the world, and it is this combinatorial complexity that allows us to perceive the breadth of smell. However, as of 2016, only 48 odorants or ligands of human ORs (about 12%) have been identified using traditional deorphanization [Teixeira CSS et al., Chemical Senses, 41(2):105-21 (2016)]. Furthermore, most of the ligands identified in vitro have not been tested for their physiological relevance to human ORs.

[0005] In WO 2014 / 210585 a method is described which enables rapid and reliable identification of a relatively small fraction of all ORs present in an organism which are specifically activated or inhibited by one or more odorants.

[0006] Because ORs are encoded by the largest gene family in vertebrates, the seven transmembrane class A G protein-coupled receptors (GPCRs), it is difficult to identify receptors that are essential for a given perception.

[0007] There remains a need to identify OR receptors that are activated by specific odorants and / or aromatic compounds. Once one or more ORs activated by a specific odorant and / or aromatic compound or a group of such compounds are identified, it is necessary to identify the relevant odorant receptors that can be used in assays and methods for identifying and pharmacologically characterizing volatile modulators, such as allosteric modulators, of OR activity. Such allosteric modulation of odorant receptor activity is speculative at best and poorly understood in the field of olfaction.

[0008] Therefore, there is a need for methods for identifying relevant receptors and compounds that can be used in perfumery or other applications to modulate, improve or enhance (using positive allosteric modulators) a desired aroma or fragrance, which can lead to a more sensitive and more intense perception of the fragrance or aroma note, and to inhibit or counteract (using negative allosteric modulators or competitive antagonists) undesirable odors or compounds. Summary of the invention

[0009] One aspect presented herein provides a high-throughput assay system for identifying one or more compounds from a group of compounds, wherein the one or more compounds positively modulate the activity of an olfactory receptor induced by an agonist odorant compound, comprising:

[0010] a. one or more isolated cells, each cell expressing a mammalian olfactory receptor, wherein the one or more cells contain a target olfactory receptor activated by one or more odorants,

[0011] b. a first compound that binds to and activates the olfactory receptor, and

[0012] c. at least one second compound that binds to the receptor non-competitively with respect to the first compound and enhances the activity of the receptor exposed to the first compound compared to the activity of the receptor exposed to the first compound in the absence of the second compound,

[0013] wherein the first compound is an agonist odorant compound of the olfactory receptor,

[0014] wherein the first compound and the second compound are different compounds,

[0015] wherein the second compound is not an agonist of the receptor, and

[0016] Wherein the activity of the receptor is synergistically enhanced by the combination of the first compound and the second compound.

[0017] In one form, the at least one second compound is selected from a group of compounds based on structure-activity relationship (SAR) analysis.

[0018] In one form, the second compound binds to a site on the olfactory receptor that is different from the site to which the first compound binds.

[0019] In one form, the second compound is a positive allosteric modulating compound.

[0020] In one aspect of the assays or methods herein, the olfactory receptor is selected from the group consisting of OR5AN1, Olfr1440, OR10J5, or Olfr16.

[0021] In one aspect of the assays or methods herein, the at least one olfactory receptor comprises a polypeptide that:

[0022] a. comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2, 4, 6 or 8; and / or

[0023] b. Encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 3, 5 or 7 or its reverse complement.

[0024] In one aspect, the at least one olfactory receptor comprises a polypeptide that:

[0025] a. comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, 4, 6 or 8; and / or

[0026] b. Encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 3, 5 or 7 or its reverse complement.

[0027] In one form of the assay or method, the first compound is a musk or muguet compound.

[0028] In one form, the mammalian olfactory receptor is a mouse olfactory receptor or a human olfactory receptor.

[0029] In one embodiment, the first compound comprises a musk compound and the olfactory receptor is OR5AN1 or Olfr1440; chimeras and functional fragments thereof; and wherein the olfactory receptor comprises a polypeptide that:

[0030] a. comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or 6; and / or

[0031] b. Encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1 or 5 or its reverse complement.

[0032] In one form, the musk compound comprises a macroketone or a nitromusk compound.

[0033] In one embodiment, the receptor is OR5AN1, the agonist odorant compound comprises a macroketone or a nitromusk compound; and the selected compound comprises at least one positive allosteric modulator having the structure:

[0034]

[0035] The compound is in the form of any one of its stereoisomers or a mixture of these stereoisomers,

[0036] Wherein R represents optionally substituted with C 1-4 Alkyl carboxyl ester or hydroxyl C 3-11 Straight chain alkyl, optionally substituted with C 1-3 Alkoxy substituted C 4-11 Branched alkyl, C 6-12 Straight or branched alkenyl or diene, substituted with one or two C 1-3 Alkyl phenyl, C 5-8 alicyclic alkenyl, or benzyloxymethyl, and

[0037] The compound of formula (I) is a positive allosteric modulator of musk olfactory receptor.

[0038] In another embodiment, the compound of formula (I) is selected from the group consisting of (E,E)-2,4-decadienal, (E,E)-2-undecenal, (E,E)-2,4-nonadienal, (E)-2-tridecenal, (2E)-2-dodecenal, (2E,6Z)-2,6-nonadienal, (E,E)-2,6-nonadienal, (2E)-2,4-undecadienal, (2E)-2,4-dodecadienal, (E)-2-nonenal, (2E,4E,7Z)-decatrienal, (Z)-2-decenal, (E)-2-octenal, (E)-2-decenal, (Z)-4-(benzyloxy)but-2-enal, (E) -4-(benzyloxy)but-2-enal, (E)-4-cyclohexylidenebut-2-enal, (2E)-3-(4-methylphenyl)-2-propenal, (2E,5E)-6,10-dimethyl-2,5,9-undecatrienal, (2E)-7,8-dimethyl-2,7-nonadienylcyclohexyl, (5E)-7-oxo-5-heptenoic acid methyl ester, (2E)-7-methyl-2,6-octadienal, (2E)-6,6-dimethyl-2-heptenal, (+-)-(2E)-5,9-dimethyl-2,8-decadienal, (E)-2-tetradecenal, and (E)-8-methoxy-4,8-dimethylnon-2-enal, and combinations thereof.

[0039] In another embodiment, the first compound comprises a lily of the valley compound, and the olfactory receptor is OR10J5 or Olfr16; chimeras and functional fragments thereof; and wherein the olfactory receptor comprises a polypeptide that:

[0040] a. comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4 or 8; and / or

[0041] b. Encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 3 or 7 or its reverse complement.

[0042] In one embodiment, the lily of the valley compound is 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde, 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde, or a mixture thereof.

[0043] In one embodiment, the receptor is OR10J5, the agonist odorant compound comprises a lily of the valley compound; and the selected compound comprises at least one positive allosteric modulator selected from the group consisting of: (E)-dec-2-enal, 2-phenylpropanal; (E)-but-2-enal; 3-methylbenzaldehyde; 3-(1,3-benzodioxazol-5-yl)-2-methylpropanal; (+-)-3-(4-methyl-3-penten-1-yl)-3-cyclohexene-1-carboxaldehyde, (+-)-4-(4-methyl-3-penten-1-yl)-3-cyclohexene-1-carboxaldehyde, and mixtures thereof; heptaldehyde, 4-propan-2-ylbenzaldehyde; (3 R)-3,7-dimethyloct-6-enal; (+)-(3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal, (+)-(3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal, and mixtures thereof; hexanal; 2,6-dimethylhept-5-enal; benzaldehyde; 2-methyl-3-(4-methylphenyl)propanal; 3,5,6-trimethyl-3-cyclohexene-1-carboxaldehyde, 2,4,6-trimethyl-3-cyclohexene-1-carboxaldehyde, and mixtures thereof; 4-ethylbenzaldehyde; 6-methoxy-2,6-dimethylheptanal; (E)-non-2-enal; and combinations thereof.

[0044] In one form, the modulated activity includes:

[0045] a. modulating the binding affinity of the first compound to the orthosteric site,

[0046] b. modulating the binding activation efficacy of the first compound to the orthosteric site, and / or

[0047] c. Regulate G protein binding sites.

[0048] One aspect presented herein provides a method for identifying at least one positive allosteric modulator compound that enhances the activity of an olfactory receptor in the presence of an agonist of the olfactory receptor, the method comprising:

[0049] a. providing one or more isolated cells, each cell expressing a mammalian olfactory receptor,

[0050] b. exposing one or more cells to a first compound capable of activating the olfactory receptor,

[0051] c. exposing one or more cells to one or more test compounds, and

[0052] d. selecting the at least one second compound from the one or more test compounds when the at least one second compound has a synergistic effect on receptor activity in combination with the first compound as a positive allosteric modulator;

[0053] wherein the first compound is an agonist odorant compound of the olfactory receptor,

[0054] wherein the first compound and the second compound are different compounds, and

[0055] wherein the second compound is not an agonist of the receptor.

[0056] In one aspect, activation of the olfactory receptor to a first compound is measured by measuring the response of the olfactory receptor in the presence and absence of the first compound.

[0057] In one form, at least one second compound is selected from one or more test compounds in a chemically diverse library of compounds.

[0058] In one aspect, at least one second compound is selected from the one or more test compounds based on a structure-activity relationship (SAR) analysis of the initially identified compound.

[0059] The method of any of the preceding aspects, wherein the second compound binds to a site on the olfactory receptor that is different from the site to which the first compound binds.

[0060] In one aspect, prior to step a) of the assay systems or methods provided herein, the one or more cells are transformed to express a target olfactory receptor.

[0061] In one form, the olfactory receptor is heterologous to the one or more cells.

[0062] In one form, the cell is selected from the group consisting of HEK293, CHO, Xenopus oocytes, COS, insect, yeast, and cells derived from the olfactory placode.

[0063] One embodiment presented herein provides a method for identifying a compound that enhances the activity of an olfactory receptor induced by an agonist odorant compound, the method comprising:

[0064] (i) screening one or more compounds in a binding assay in the presence of an agonist odorant compound;

[0065] (ii) selecting one or more screened compounds that can specifically enhance the specific binding or binding effect of the agonist odorant compound to the olfactory receptor of a mammal; and

[0066] (iii) identifying compounds that potentially modulate the perception of an agonist odorant compound based on its specific binding or enhancement of the binding effect to the olfactory receptor.

[0067] In one aspect, the enhancement of the binding of the agonist odorant compound to the olfactory receptor comprises enhancing the potency and / or efficacy of the activity of the receptor as compared to the activity of the receptor in the absence of the selected compound.

[0068] In one aspect, a selected compound that enhances the potency and / or efficacy of the activity of the receptor as compared to the activity of the receptor in the absence of the selected compound can be covalently bound to the target receptor.

[0069] In one embodiment, the receptor is OR5AN1, the agonist odorant compound comprises a musk compound; and the selected compound is a positive allosteric modulator, comprising a positive allosteric modulator having the structure:

[0070]

[0071] The compound is in the form of any one of its stereoisomers or a mixture of these stereoisomers,

[0072] Wherein R represents optionally substituted with C 1-4 Alkyl carboxyl ester or hydroxyl C 3-11 Straight chain alkyl, optionally substituted with C 1-3 Alkoxy substituted C 4-11 Branched alkyl, C 6-12 Straight or branched alkenyl or diene, substituted with one or two C 1-3 Alkyl phenyl, C 5-8 alicyclic alkenyl, or benzyloxymethyl, and

[0073] The compound of formula (I) is a positive allosteric modulator of musk olfactory receptor.

[0074] In one embodiment, the compound of formula (I) is selected from the group consisting of: (E,E)-2,4-decadienal, (E,E)-2-undecenal, (E,E)-2,4-nonadienal, (E)-2-tridecenal, (2E)-2-dodecenal, (2E,6Z)-2,6-nonadienal, (E,E)-2,6-nonadienal, (2E)-2,4-undecadienal, (2E)-2,4-dodecadienal, (E)-2-nonenal, (2E,4E,7Z)-decatrienal, (Z)-2-decenal, (E)-2-octenal, (E)-2-decenal, (Z)-4-(benzyloxy)but-2-enal , (E)-4-(benzyloxy)but-2-enal, (E)-4-cyclohexylidenebut-2-enal, (2E)-3-(4-methylphenyl)-2-propenal, (2E,5E)-6,10-dimethyl-2,5,9-undecatrienal, (2E)-7,8-dimethyl-2,7-nonadienylcyclohexyl, (5E)-7-oxo-5-heptenoic acid methyl ester, (2E)-7-methyl-2,6-octadienal, (2E)-6,6-dimethyl-2-heptenal, (+-)-(2E)-5,9-dimethyl-2,8-decadienal, (E)-2-tetradecenal, and (E)-8-methoxy-4,8-dimethylnon-2-enal.

[0075] In one embodiment, the receptor is OR10J5, the agonist odorant compound comprises a lily of the valley compound; and the selected compound comprises at least one positive allosteric modulator selected from the group consisting of: octanal; (E)-dec-2-enal, 2-phenylpropanal; (E)-but-2-enal; 3-methylbenzaldehyde; 3-(1,3-benzodioxazol-5-yl)-2-methylpropanal; (+-)-3-(4-methyl-3-penten-1-yl)-3-cyclohexene-1-carboxaldehyde, (+-)-4-(4-methyl-3-penten-1-yl)-3-cyclohexene-1-carboxaldehyde, and mixtures thereof; heptaldehyde, 4-propan-2-ylbenzaldehyde; ( 3R)-3,7-dimethyloct-6-enal; (+)-(3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal, (+)-(3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal, and mixtures thereof; hexanal; 2,6-dimethylhept-5-enal; benzaldehyde; 2-methyl-3-(4-methylphenyl)propanal; 3,5,6-trimethyl-3-cyclohexene-1-carboxaldehyde, 2,4,6-trimethyl-3-cyclohexene-1-carboxaldehyde, and mixtures thereof; 4-ethylbenzaldehyde; 6-methoxy-2,6-dimethylheptanal; (E)-non-2-enal; and combinations thereof.

[0076] In one aspect, one or more selected compounds are selected based on a structure-activity relationship (SAR) analysis specifically designed to identify putative chemical determinants of potentiation.

[0077] In one form, screening involves calculating an activation window to confirm the statistical quality of the data and measuring the 05-50 In another form, in EC 20 The synergistic effect is enhanced.

[0078] One aspect presented herein provides the use of a polypeptide activated by an agonist, the polypeptide being selected from a human olfactory receptor activated by the agonist, in a screening assay for identifying one or more positive allosteric modulatory compounds of the agonist.

[0079] One embodiment presented herein provides the use of a polypeptide that can be activated by musk, which comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 or 6, for identifying positive allosteric modulatory compounds of musk.

[0080] One embodiment presented herein provides the use of a polypeptide that can be activated by a lily of the valley compound, wherein the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4 or 8, for identifying a positive allosteric modulator compound of the lily of the valley receptor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Figure 2 shows the number of musk-induced in vitro OR5AN1 activation levels for a series of different musks (a) and the number of odor detection threshold records for muscone (3-methyl-cyclopentadecanone), Correlation between the overall enhanced human sensitivity of musk xylene and musk ketone (b).

[0082] Figure 2 A method for screening OR5AN1 for musk-induced enhancement of activity is shown. Results of single-site binary mixtures with fragrance raw materials.

[0083] FIG3 shows the effects of OR5AN1 on all four agonists muscone, Dose response curves for musk xylene and musk ketone, and the dose response to tridecenal alone.

[0084] Figure 4 The EC obtained from the dose-response curves obtained in Figure 3 are given 50 The values ​​were plotted and the corresponding EC50 fold shifts were summarized.

[0085] Figure 5 shows the effect of OR5AN1 on A series of dose-response curves are shown, and the synergistic factor α and the equilibrium constant K are also indicated. B The calculated logarithm of .

[0086] Figure 6 A ranking of 67 volatile compounds based on potency shift and efficacy increase according to their OR5AN1 musk response enhancing abilities is shown.

[0087] Figure 7 Delineated are the chemical requirements identified from the SAR analysis that preferentially elicit an enhanced OR5AN1 musk response.

[0088] FIG8 shows the different enhancement levels obtained on OR5AN1 using a) decenal, b) decadienal and c) decanal, respectively, and the requirements for the degree of unsaturation.

[0089] Figure 9 shows the different enhancement levels obtained on OR5AN1 using a) dodecenal and b) tangerinal, respectively, and the importance of the position of unsaturation.

[0090] FIG. 10 shows the different enhancement levels obtained on OR5AN1 using a) nonenal, b) nonenol and c) nordecenol, respectively, and in the absence of enhancement via the alcohol functionality.

[0091] FIG. 11 shows the different enhancement levels obtained on OR5AN1 using a) decenal and b) decenone, respectively, and in the absence of enhancement via the ketone functionality.

[0092] Figure 12 shows the different enhancement levels obtained on OR5AN1 using a) undecenal and b) undecylenic acid, respectively, and in the absence of enhancement by the acid group.

[0093] Figure 13 shows the When activated, OR5AN1 alleles are functionally unequal, but enhancement restores the activity of the functionally deficient allele.

[0094] Fig.14 shows that human panelists responded to More sensitive.

[0095] Fig.15 Shown is the dose response curve of OR10J5 to the floral lily of the valley compound.

[0096] Fig.16 The floral lily of the valley compound 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carboxaldehyde, 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carboxaldehyde or a mixture thereof (trade name Sale (hereinafter referred to as ) and the results of single-site binary mixtures used to screen for OR10J5-induced enhanced activity of fragrance raw materials.

[0097] Figure 17 shows the OR10J5 Different levels of enhancement obtained under induced activation using a) heliopropanal, b) 3-(4-methylcyclohexyl-3-en-1-yl)butanal, c) heliotropin and d) cyclomethylenecitronellol, with the absence of enhancement by the latter two compounds.

[0098] Figure 18 shows the Different levels of enhancement obtained under induced activation using a) heliotropin, b) 3-(4-methylcyclohexyl-3-en-1-yl)butanal, c) heliotropin and d) cyclomethylenecitronellol, respectively, with the absence of enhancement by the latter two compounds.

[0099] FIG. 19 shows the effect of OR10J5 on the performance of the hydroxyl radical in the presence of a) hydroxyl radical, b) 3-(4-methylcyclohexyl-3-en-1-yl)butyraldehyde, c) melonaldehyde, and d) heptaldehyde in the presence of a range of concentrations. A series of dose-response curves are shown, and the synergistic factor α and the equilibrium constant K are also indicated. B The corresponding calculated logarithm of .

[0100] Fig. 20 shows that human panelists responded to More sensitive.

[0101] Fig.21 shows the increase in the number of human panelists according to the concentration of the enhancer compared to the non-enhancing compound Detection quantity.

[0102] FIG. 22 shows the different levels of enhancement obtained upon activation induced by OR10J 5(+-)-2,5-dimethyl-2-indanemethanol, using a) heliotropin, and b) heliotropin, respectively, and the absence of enhancement by the latter compound. DETAILED DESCRIPTION

[0103] definition

[0104] For purposes of this document and the appended claims, the use of "or" means "and / or" unless stated otherwise.

[0105] Similarly, the various tenses of "comprising," "including," "comprising," and "including" are interchangeable and not limiting.

[0106] It should be further understood that where the term “comprising” is used in the description of various embodiments, those skilled in the art will appreciate that in some specific cases, the language “consisting essentially of” or “consisting of” may be used to alternatively describe the embodiments.

[0107] Unless otherwise specified, the following terms have the meanings ascribed to them.

[0108] As used herein, the term "OR" or "olfactory receptor" or "odorant receptor" refers to one or more members of the G protein-coupled receptor (GPCR) family expressed in olfactory cells. Olfactory receptor cells can also be identified based on morphology or by expression of proteins specifically expressed in olfactory cells. OR family members may have the ability to act as receptors for olfactory signal transduction.

[0109] A target olfactory receptor is an olfactory receptor that can be associated with sensory perception in vitro, in vivo, or ex vivo.

[0110] "Musk OR" refers to a member of the G protein-coupled receptor (GPCR) family expressed in olfactory cells, which receptor binds to and / or is activated by musk in a binding or activity assay for identifying ligands that bind and / or activate GPCRs. Such assays are described below. The musk receptors herein will include fragments, variants, both synthetic and naturally occurring, as well as chimeras or recombinant nucleic acids or proteins that correspond or bind to musk compounds. The same definition applies similarly to other related or target ORs that can be identified by the methods described herein.

[0111] Odorants refer to any type of odorants, including but not limited to fragrances (daily chemical flavors), flavor ingredients, aromatics, foods, seasonings (edible flavors) or seasoning ingredients.

[0112] An agonist or agonist compound of an OR refers to a compound or ligand that binds to an OR, activates the OR and has the potential to induce the receptor transduction cascade.

[0113] As contemplated herein, a compound that is not an agonist of an olfactory receptor refers to a compound or ligand that binds to the OR non-competitively relative to an agonist compound and enhances or inhibits the activity of the receptor exposed to the agonist compound when compared to the activity of the receptor exposed to the agonist compound in the absence of the non-agonist compound. In one form, a compound that is not an agonist binds to a site on the receptor that is different from the site to which the agonist compound binds.

[0114] In one form, the potency and efficacy of a compound that is not an olfactory receptor agonist is significantly lower than that of an agonist, e.g., when EC 50 Values ​​are significantly higher than the potency of agonist compounds and the potency is generally less than 20% of the potency of a full agonist response at saturation.

[0115] Potency is a measure of the receptor activity induced by the binding of an agonist (odorant) in terms of the amount required to produce a given level of activity. It expresses the sensitivity of the receptor to different agonist concentrations and is usually calculated by calculating the EC 50 (the concentration of agonist necessary to achieve half-maximal receptor activity) was obtained.

[0116] Efficacy is a measure of how strongly a receptor responds to a given agonist and is obtained by measuring the range of activation between constitutive (baseline activity in the absence of agonist) and agonist-induced activity. Maximum efficacy is achieved when the receptor is saturated.

[0117] The orthosteric binding site refers to the site where a ligand or odorant compound binds to an agonist or competitive antagonist. Binding events to the orthosteric site typically result in activation of the agonist or inhibition of receptor activity by a competitive antagonist.

[0118] An allosteric binding site is a binding site that is topologically different from an orthosteric binding site. Allosteric binding in the presence of an orthosteric agonist is not sterically hindered and therefore occurs in a non-competitive manner. Certain allosteric ligands can act as positive allosteric modulators (PAMs) or negative allosteric modulators (NAMs), for example to enhance or inhibit, respectively, activation of a receptor by an agonist.

[0119] Binding competition occurs when two molecules compete to bind to the same binding site. The binding probability of each ligand depends on its affinity to the receptor and its concentration compared to the other ligands. Competition usually occurs when both ligands bind at the orthosteric site. This can be identified by performing a competition assay (e.g., Schild regression analysis). When no competition is observed, two ligands can bind to the receptor simultaneously at two different binding sites (defined as orthosteric and allosteric binding sites), where the binding sites can be topologically separated, adjacent, or overlapping on the receptor. In this case, the binding of the orthosteric ligand is not linearly dependent on the concentration of the allosteric ligand. Allosteric binding can modulate some properties of the receptor, such as agonist affinity and signal transduction efficiency.

[0120] A positive or negative allosteric modulator or a positive or negative allosteric modulator compound refers to a compound or ligand that enhances or inhibits the effect of orthosteric agonist compound or ligand binding but is substantially inactive in the absence of the orthosteric agonist compound or ligand. Allosteric inhibition or enhancement of receptor activity can be performed by modulating the conformation of the receptor and modifying: 1) orthosteric site affinity for agonists; 2) the effect (e.g., efficacy) of orthosteric binding activation; or 3) in the case of GPCRs, binding to G proteins, thereby reducing or increasing signal transduction efficiency, respectively.

[0121] The synergistic factor is a measure of the degree to which the binding of an orthosteric ligand is modulated by the presence of an allosteric ligand.

[0122] The ability of the disclosed compounds to inhibit or antagonize olfactory receptors can be determined by any suitable method readily selected by one of ordinary skill in the art, for example, by an in vitro cultured neuron assay, or by an in vivo assay using a cell line expressing the relevant olfactory receptor (e.g., the olfactory receptor for musk).

[0123] Such assays for inhibitors and activators include, for example, expressing OR family members in cells or cell membranes with putative modulator compounds in the presence or absence of agonist molecules (e.g., musk), and then determining the functional effects on olfactory transduction, as described in the following examples. Samples or assays containing OR family members treated with potential inhibitors are compared with control samples without inhibitors to examine the degree of inhibition. The relative maximum OR activity value of the control sample (not treated with inhibitors, but treated with agonists) is designated as 100%.

[0124] Olfactory receptor activity assays can reveal the following data: (i) whether a given compound is an activator of an olfactory receptor and how specific the compound is for a particular olfactory receptor; (ii) the EC value of the agonist for the olfactory receptor 50 (i.e., the EC of the agonist 50); and / or (iii) the efficacy of an agonist at an olfactory receptor, which depends on the amplitude of the response (i.e., the span between baseline and saturation activity levels).

[0125] In some embodiments, an enhancement of an OR is achieved when the normalized OR activity value relative to the agonist control (100%) is greater than 100%, such as about 110%, optionally 120% or 150% or greater. In one embodiment, an enhancement of an OR is achieved if the potency of an agonist on an OR is increased in the presence of an enhancer compound. In one embodiment, an enhancement of an OR is achieved by an agonist's EC 50 Alternatively, in another form, if the EC of the agonist in the presence of the enhancer compound 50 The enhancement of OR is achieved if the EC of the agonist compound is reduced by a factor of 2 to 30. 50 In another embodiment, if the EC of the agonist in the presence of the enhancer compound is reduced by a factor of two, then enhancement of the OR is achieved. 50 If the value is reduced to one-thirtieth, an enhancement of OR is achieved.

[0126] In some embodiments, enhancement of an OR can be achieved if the efficacy of an OR agonist in the presence of an enhancer compound is increased relative to an agonist control. In one embodiment, enhancement of an OR is achieved if the efficacy value of the agonist in the presence of an enhancer compound is increased by 1.05-fold to 2-fold or more. In one embodiment, enhancement of an OR is achieved if the efficacy value of the agonist in the presence of an enhancer compound is increased by about 1.05-fold. In another embodiment, enhancement of an OR is achieved if the efficacy value of the agonist in the presence of an enhancer compound is increased by about 2-fold.

[0127] In one embodiment, inhibition of an OR is achieved when the normalized OR activity value relative to the agonist control (100%) is less than 100%, for example, about 80%, and preferably 50% or 25-0%. In one embodiment, inhibition of an OR is achieved if the potency of the agonist for the OR is reduced. In another embodiment, inhibition of an OR is achieved by the EC of the agonist. 50 In one form, if the EC of an agonist in the presence of an antagonist compound is 50 Inhibition of the OR is achieved if the EC of the agonist in the presence of the antagonist compound is increased by about 2-fold to about 30-fold. 50 Inhibition of the OR is achieved if the EC of the agonist is increased by 2-fold in the presence of the antagonist compound. 50 When the value increases to 30 times, the inhibition of OR is achieved.

[0128] In some forms, if the efficacy of an OR agonist is reduced in the presence of an antagonist compound, inhibition of an OR can be achieved. In one form, if the efficacy value of the agonist in the presence of an antagonist compound is reduced to about 1.25 to 1 / 4 or less, inhibition of an OR is achieved. In one form, if the efficacy value of the agonist in the presence of an antagonist compound is reduced to 0, inhibition of an OR is achieved. In another form, if the efficacy value of the agonist in the presence of an antagonist compound is reduced to 1.25, inhibition of an OR is achieved. In one form, if the efficacy value of the agonist in the presence of an antagonist compound is reduced to 1 / 4, inhibition of an OR is achieved.

[0129] Synergistically enhanced activity of an OR refers to an increase in the activity of the receptor by exposure of the receptor when the combination of an agonist compound and a positive allosteric modulator compound is present, compared to the activity of the receptor when exposed to an agonist alone. In one form, the synergistically enhanced activity includes an increase in potency of about 2-fold to 30-fold and / or an increase in efficacy of about 1.05-fold to 1.5-fold or more.

[0130] "OR" or "odorant receptor" or "olfactory receptor" polypeptides are considered to be such that they belong to the 7-transmembrane domain G protein-coupled receptor superfamily encoded by a single ~1 kb long exon and exhibit characteristic olfactory receptor-specific amino acid motifs. These seven domains are referred to as "transmembrane" or "TM" domains TM I to TM VII, which are connected by three "inner cell loop" or "IC" domains IC I to IC III, and three "outer cell loop" or "EC" domains EC I to EC III. The motif and its variants are defined as, but not limited to, the MAYDRYVAIC motif (SEQ ID NO: 15) overlapping with TM III and IC II, the FSTCSSH motif (SEQ ID NO: 16) overlapping with IC III and TM VI, the PMLNPFIY motif (SEQ ID NO: 17) in TM VII, and the three conserved C residues in EC II, and the presence of the highly conserved GN residue in TM I [Zhang, X. & Firestein, S. Nat. Neurosci. 5, 124–133 (2002); Malnic, B., et al. Proc. Natl. Acad. Sci. USA 101, 2584–2589 (2004)].

[0131] "OR" nucleic acids encode a family of GPCRs with seven transmembrane domains that have "G protein-coupled receptor activity", i.e., they can bind G proteins in response to extracellular stimuli and promote the production of second messengers such as IP by stimulating enzymes such as phospholipase C and adenylate cyclase. 3 , cAMP, cGMP and Ca2+ The production of.

[0132] "Paralogous" OR genes or "paralogs" are the result of gene duplication and refer to closely related homologous genes within the same species.

[0133] "Orthologous" OR genes or "orthologs" are defined as being phylogenetically connected by genes present in a common ancestor, and refer to closely related homologous genes in other species.

[0134] The "N-terminal domain" region begins at the N-terminus (amino terminus) of a peptide or protein and extends to a region near the start of the first transmembrane region.

[0135] "Spanning membrane region" includes seven "spanning membrane domains", which refers to the domain of the OR polypeptide located in the plasma membrane, and may also include corresponding cytoplasmic loops (intracellular) and extracellular loops. Seven transmembrane regions, extracellular loops and cytoplasmic loops can be identified using standard methods, such as hydrophobicity profiles, or as described in Kyte & Doolittle, J. Mol. Biol., 157: 105-32 (1982) or in Stryer. The general secondary and tertiary structures of transmembrane domains, particularly the seven transmembrane domains of G protein coupled receptors, such as olfactory receptors are known in the art. Therefore, the primary structure sequence can be predicted based on known transmembrane domain sequences, as described in detail below. These transmembrane domains can be used for in vitro ligand binding assays.

[0136] In the context of assays testing compounds that modulate olfactory transduction mediated by OR family members, the phrase "functional effect" includes determining any parameter that is indirectly or directly affected by the receptor, such as functional, physical, and chemical effects. It includes ligand binding, ion flux, membrane potential, current, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, signal transduction receptor-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP IP 3 or intracellular Ca 2+ ) in vitro, in vivo and ex vivo, and also include other physiological effects, such as increases or decreases in the release of neurotransmitters or hormones.

[0137] In the context of an assay, "determining a functional effect" or "confirming activity" refers to the determination of a compound that increases or decreases a parameter that is indirectly or directly affected by an OR family member, such as a functional, physical, and chemical effect. These functional effects can be measured by any method known to those skilled in the art, for example, spectral characteristics (e.g., fluorescence, absorbance, refractive index), fluid dynamics (e.g., shape), chromatographic or solubility properties, patch clamp, voltage-sensitive dyes, whole-cell currents, radioisotope efflux, inducible labeling, changes in oocyte OR gene expression; OR expression in tissue culture cells; transcriptional activation of OR genes or activity-induced genes (such as egr-1 or c-fos); ligand binding assays; changes in voltage, membrane potential, and conductance; ion flux assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, etc.

[0138] As used herein, the terms "purified", "substantially purified" and "isolated" refer to a state free of other different compounds that are normally associated with the compound of the invention in its natural state, so that the "purified", "substantially purified" and "isolated" articles account for at least 0.5%, 1%, 5%, 10% or 20% by weight, or at least 50% or 75% by weight of the mass of a given sample. In a specific embodiment, these terms refer to the compound of the invention accounting for at least 95%, 96%, 97%, 98%, 99% or 100% by weight of the mass of a given sample. As used herein, when referring to nucleic acids or proteins, the terms "purified", "substantially purified" and "isolated" of nucleic acids or proteins also refer to a state of purification or concentration different from that of the nucleic acid or protein naturally present in mammals, especially humans. Any degree of purification or concentration, as long as it is greater than the purity or concentration naturally present in mammals, especially humans, including (1) purification from other related structures or compounds, or (2) association with structures or compounds that are not normally associated in mammals, especially humans, is within the meaning of "isolated". The nucleic acids, proteins or species of nucleic acids or proteins described herein may be isolated or otherwise associated with structures or compounds to which they are not normally related in nature according to a variety of methods and processes known to those skilled in the art.

[0139] As used herein, the terms "amplifying" and "amplification" refer to the use of any suitable amplification method for producing or detecting recombinant naturally expressed nucleic acids, as described in detail below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo dT primers) for amplifying (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic DNA or mRNA) or recombinant nucleic acids (e.g., cDNA) of the invention in vivo, ex vivo or in vitro.

[0140] The term "7-transmembrane receptor" refers to a polypeptide belonging to the transmembrane protein superfamily, which has seven domains that span the plasma membrane seven times (thus, these seven domains are referred to as "transmembrane" or "TM" domains TM I to TM VII). The family of olfactory and certain taste receptors belong to this superfamily. 7-transmembrane receptor polypeptides have similar and characteristic primary, secondary and tertiary structures, as discussed in further detail below.

[0141] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in single-stranded or double-stranded form. The term includes nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also includes nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating, for example, sequences in which the third position of one or more selected codons is substituted with mixed bases and / or deoxyinosine residues.

[0142] In addition to the gene sequences shown in the sequences disclosed herein, variants also include DNA sequence polymorphisms that may exist in a given population, which may result in changes in the amino acid sequence of the polypeptide disclosed in the present invention, which is obvious to those skilled in the art. Due to natural allelic variation, this genetic polymorphism may exist in cells from different populations or in cells from a population. Allelic variants may also include functional equivalents.

[0143] Further embodiments also relate to molecules derived from such sequence polymorphisms of the specifically disclosed nucleic acids. These natural variations generally produce about 1 to 5% changes in the nucleotide sequence of a gene or the amino acid sequence of a polypeptide disclosed herein. As described above, nucleic acids encoding polypeptides of the embodiments herein are useful tools for modifying non-human host organisms or host cells intended for use in the methods described herein.

[0144] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, whether or not comprising naturally occurring amino acids or polymers and non-naturally occurring amino acids or polymers. When used with reference to a portion of a nucleic acid, the term "heterologous" means that the nucleic acid comprises two or more subsequences that do not exist in the same relationship to each other in nature. For example, nucleic acids are typically recombinantly produced, with two or more sequences from unrelated genes that are arranged to produce a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that do not exist in the same relationship to each other in nature (e.g., a fusion protein).

[0145] "Promoter" is defined as an array of nucleic acid sequences that directs the transcription of nucleic acids. As used herein, a promoter includes a TATA box of necessary nucleic acid sequences near the transcription start site, for example, in the case of a polymerase II type promoter. The promoter may also optionally include a distal enhancer or repressor element, which may be located several thousand base pairs from the transcription start site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably connected" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter or transcription factor binding site array) and a second nucleic acid sequence, wherein the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0146] As used herein, "recombination" refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., a "recombinant polynucleotide"); refers to a method of using a recombinant polynucleotide to produce a gene product in a cell or other biological system; or refers to a polypeptide encoded by a recombinant polynucleotide ("recombinant protein"). "Recombination" also means connecting nucleic acids having various coding regions or domains or promoter sequences from different sources to an expression cassette or vector for expression, for example, of an inducible or constitutive expression of a fusion protein comprising a translocation domain of the present invention and a nucleic acid sequence potentially amplified using primers. "Recombination" also refers to the modification of cells resulting in stable or transient expression of endogenous genes (e.g., receptor genes mentioned herein) obtained by genome editing techniques (e.g., CRISPR / Cas9).

[0147] The term "expression vector" refers to any recombinant expression system for expressing the nucleic acid sequence of the present invention in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cells, in vitro, in vitro or in vivo, constitutively or inducibly. The term includes any linear or circular expression system, including but not limited to viral vectors, phages and plasmids. The technician can select a suitable vector according to the expression system. The term includes expression systems that remain episomal or integrated into the host cell genome. The expression system can have the ability to self-replicate or non-self-replicate, i.e., drive transient expression in cells. The term includes recombinant "expression cassettes", which may contain the minimum elements required for transcribing recombinant nucleic acids. The term also encompasses boxes or vectors for expressing endogenous genes by, for example, genome editing methods (e.g., CRISPR / Cas9).

[0148] "Non-human organism or host cell" refers to a non-human organism or cell that contains a nucleic acid or expression vector described herein and supports replication or expression of the expression vector. The host cell can be a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as yeast, insect, amphibian or mammalian cell, such as CHO, HeLa, HEK-293, etc., such as cultured cells, explants, and in vivo cells.

[0149] A "tag" or "tag combination" refers to a short polypeptide sequence that can be added to an odorant receptor protein. Typically, the DNA encoding the "tag" or "tag combination" is added to the DNA encoding the receptor, ultimately producing a fusion protein in which the "tag" or "tag combination" is fused to the N-terminus or C-terminus of the receptor. Lucy, And / or Rho tags can enhance receptor trafficking to the cell membrane and therefore can help express functional odorant receptors for in vitro cell-based assays [Shepard, B. et al. PLoS One 8, e68758–e68758 (2013), and Zhuang, H. & Matsunami, HJ Biol. Chem. 282, 15284–15293 (2007)].

[0150] Specific Implementation

[0151] The assays and methods described herein utilize the relevant human olfactory receptors and / or corresponding mouse orthologs in assays and methods for identifying one or more compounds from a panel of compounds, wherein the one or more compounds positively modulate the activity of the olfactory receptor, which is induced by an agonist odorant compound, wherein the agonist is of particular interest to the perfume industry, and uses a combination of the target receptor, its agonist and the one or more compounds in a screening assay or method.

[0152] The present disclosure provides identification of compounds that can be used in perfumery applications to modulate, i.e., enhance, respectively, a desired aroma or target agonist, such as a musk or floral compound, such as a muscone or floral lily of the valley compound. In some forms, the volatile compound that enhances the target agonist is a positive allosteric modulator of the target agonist olfactory receptor. Without being limited to any particular theory, synergistic interactions between perfume ingredients can result in a more sensitive and more intense perception of perfume tonality. Such compounds can therefore provide new approaches to optimize perfume rules and produce better performing commercial perfume formulations.

[0153] In one embodiment, human olfactory receptor OR5AN1 and / or its corresponding mouse ortholog Olfr1440 are used as receptors activated by various musk agonist compounds to identify compounds that modulate receptor activity in the presence of musk agonist compounds. Human olfactory receptor OR5AN1 and its corresponding mouse ortholog Olfr1440 have about 66% sequence identity at the amino acid level.

[0154] In one embodiment, given the correlation between sensory outcomes and the response of OR5AN1 to musk compounds, OR5AN1 was selected as a relevant target receptor activated by specific musks, namely macroketones and nitromusks, for use in assays and methods for screening and selecting positive allosteric modulators of these musk compounds.

[0155] In another embodiment, human olfactory receptor OR10J5 and / or its corresponding mouse ortholog Olfr16 are used as receptors for various lily of the valley agonist compounds to identify compounds that modulate receptor activity in the presence of lily of the valley agonist compounds. Human olfactory receptor OR10J5 and its corresponding mouse ortholog Olfr16 have approximately 87% sequence identity at the amino acid level.

[0156] The activity induced by the floral lily of the valley compounds of OR10J5 and its corresponding mouse ortholog Olfr16 was associated with sensory outcomes of the perception of floral, lily of the valley, hydroxycitronellal odor notes and an overall olfactory profile strongly reminiscent of the well-known ingredients 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde, 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde (trade name For sale, hereinafter referred to as )'s fragrance.

[0157] Floral lily of the valley compounds refer to certain compounds that impart lily of the valley-like or muguet floral notes, including but not limited to lyral compounds.

[0158] Examples of floral lily of the valley compounds include, but are not limited to, International Patent Application Publication Nos. WO2017 / 009175 A1 or WO2010 / 091969 A1 or WO2011 / 029743 A1 or WO2018 / 134221 A1 or WO2008 / 068310 A1 or WO2014 / 198709 A1 or WO2013 / 117433 A1 or WO2014 / 180945 A1 or WO2014 / 180952 A1 or WO2016 / 074118 A1 or WO2016 / 074697 A1 or WO2016 / 074719 A1 or WO2014 / 180952 A1, or WO2001 / 090038 A1, or WO2008 / 053148 A1. A1, or the compound disclosed in WO2017 / 066214 A1.

[0159] Other examples of lily of the valley compounds include, but are not limited to, compounds disclosed in US Patent Application Publication Nos. US2013 / 0090390A1 or US2011 / 0117046 A1 or US2011 / 0118170 A1 or US2009 / 0036347 A1 or US2011 / 0217257 A1.

[0160] Other examples of lily of the valley compounds include, but are not limited to, compounds disclosed in US Pat. Nos. 5,527,769 A or 7,834,219 B1 or 2,710,825 A or 4,352,937 A.

[0161] Other examples of lily of the valley compounds include, but are not limited to, compounds disclosed in European Patent Application Publication No. EP2594626 A1 or EP0392258 A2 or EP2322495 A1 or EP1029845 A1 or EP1054053 A2 or EP1529770 A1.

[0162] Other examples of lily of the valley compounds include, but are not limited to, compounds disclosed in European Patent Nos. EP2594626 Bl or EP685444 Bl or EP2594626 Bl.

[0163] Other examples of lily of the valley compounds include, but are not limited to, compounds disclosed in British Patent Application Publication Nos. GB2528467A or GB988502A or GB1057360A or GB2529901A.

[0164] Other examples of lily-of-the-valley compounds include, but are not limited to, compounds disclosed in Lamboley et al., Synthesis and Properties of Conformationally Constrained Analogues of Floral-Type Odorants, Helvetica Chimica Acta, vol. 84, pp 1767-1793 (2004).

[0165] Other examples of lily of the valley compounds include, but are not limited to, compounds disclosed in Schroeder et al., γ-Unsaturated Aldehydes as Potential Lilial Replacers, Chemistry & Biodiversity, vol. 11, pp 1651-1673 (2014).

[0166] Other examples of lily of the valley compounds include, but are not limited to, the compounds disclosed in Skouroumounis et al., Synthesis of 1,3,4,5-Tetrahydro-2-benzoxepin Derivatives as Conformationally Restricted Analogues of Cyclamenaldehyde-Type Compounds and as Intermediates for Highly Odour-Active Homologues, Helvetica Chimica Acta, vol. 79, pp 1095-1109 (1996).

[0167] Other examples of floral lily of the valley compounds include, but are not limited to, compounds disclosed in Winter et al., Synthesis and Odor Properties of Substituted Indane-2-carboxaldehydes. Discovery of a New Floral (Muguet) Fragrance Alcohol, Helvetica Chimica Acta, vol. 88, pp3118-3127 (2005).

[0168] Other examples of floral lily of the valley compounds include, but are not limited to, compounds disclosed in Coulomb, J, Beyond Mguet, Perfume and Flavorist, vol. 43 (2018).

[0169] In some embodiments, the lily of the valley compound is selected from the group consisting of: 3(4)-(4-hydroxy-4-methylpentyl)cyclohexyl-3-ene-1-carbaldehyde, 4(4)-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde or mixtures thereof; (4Z)-9-hydroxy-5,9-dimethyl-4-decenal (A) + (4E)-9-hydroxy-5,9-dimethyl-4-decenal; 3-[4-(2-hydroxy-2-methylpropyl)-2-methylphenyl]propanal; 3-[4-(2-hydroxy-2-methylpropyl)phenyl]propanal; (+-)-3-[ 4-(2-hydroxy-2-methylpropyl)phenyl]-2-methylpropanal; (+-)-4-(4-hydroxy-4-methylpentyl)-3,6-dihydro-2H-pyran-2-carbaldehyde; (2,5-dimethyl-2-indanyl)methyl formate; (2,5-dimethyl-1,3-dihydroindan-2-yl)methanol; (2,5,6-trimethyl-1,3-dihydroindan-2-yl)methanol; (2,4,6-trimethyl-1,3-dihydroindan-2-yl)methanol; 4-methyl-2-(2-methylpropyl)oxan-4-ol; 7-hydroxy-3,7-dimethyloctanal; and mixtures thereof.

[0170] In some embodiments, the floral lily of the valley compound may include and / or may contain compounds such as 3(4)-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde, 4(4)-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde or mixtures thereof, which are sold under the following trade names: and

[0171] This approach can be used with other relevant target receptors and agonists, such as those of particular importance to the fragrance industry, to identify potential positive allosteric modulators of key agonists of these target receptors.

[0172] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 3, 5 or 7, or its reverse complement.

[0173] In one embodiment, the present invention provides an isolated nucleic acid sequence as described above, which encodes a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2, 4, 6 or 8.

[0174] In one embodiment, provided herein is an isolated polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2, 4, 6 or 8.

[0175] In one embodiment, the non-human organism or host cell is transformed to express a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, 4, 6 or 8.

[0176] In one embodiment, the non-human organism or host cell is transformed to express a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 2, 4, 6 or 8.

[0177] The present invention further provides an expression vector comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2, 4, 6 or 8.

[0178] Also provided herein are expression vectors comprising a nucleic acid comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 3, 5 or 7, or its reverse complement.

[0179] Also provided herein is an expression vector having a nucleic acid, wherein the nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 1, 3, 5 or 7 or a reverse complement thereof.

[0180] ORs may be specific for a single agonist, or they may be activated by a group of structurally different agonists or a group of agonists with similar structures. Agonists may also specifically activate one OR, or may activate multiple ORs. As a first step in performing the assays or methods described herein, a suitable receptor-agonist pair is identified and selected. In one form, such receptor-agonists are preferably particularly useful in designing a flavor composition.

[0181] For example, refer to Figure 1 and Example 1, the olfactory receptor OR5AN1 is a human olfactory receptor, and its musk-induced in vitro activity is related to the sensory results of musk. The OR5AN1 olfactory receptor is induced by musk compounds muscone, Musk xylene and musk ketone activated specifically, and the observed activity levels (i.e., potency and efficacy) correlated with individual sensory levels ( Figure 1 ). Thus, the in vitro screening assay for OR5AN1 activity has predictive power for the putative sensory outcome of the identified compounds, and OR5AN1 thus represents a good target for musk-directed receptor-based enhancement.

[0182] There is no particular limitation on the test substance to be tested in the methods and assays described herein. The test substance can be a natural substance or an artificial substance synthesized chemically or biologically. The test substance can be a single compound or a mixture of multiple compounds.

[0183] Reference Figure 6 , systematically found that α-β-monounsaturated disubstituted aliphatic aldehydes showed effective enhancement of OR5AN1 olfactory receptor activity.

[0184] Thus, one morphology proposed herein provides a positive allosteric modulator having the following structure:

[0185]

[0186] The compound is in the form of any one of its stereoisomers or a mixture of these stereoisomers,

[0187] Wherein R represents optionally substituted with C 1-4 Alkyl carboxyl ester or hydroxyl C 3-11 Straight chain alkyl, optionally substituted with C 1-3 Alkoxy substituted C 4-11 Branched alkyl, C 6-12 Straight or branched alkenyl or diene, substituted with one or two C 1-3 Alkyl phenyl, C 5-8 alicyclic alkenyl, or benzyloxymethyl, and

[0188] The compound of formula (I) is a positive allosteric modulator of musk olfactory receptor.

[0189] For the sake of clarity, by expressing "any stereoisomer thereof" or similar terms, the normal meaning understood by those skilled in the art is intended, i.e., the compounds of the invention may be pure enantiomers (if chiral) or diastereomers (e.g., the configuration of the double bond is E or Z).

[0190] In some embodiments, the compound of formula (I) is selected from the group consisting of (E,E)-2,4-decadienal, (E,E)-2-undecenal, (E,E)-2,4-nonadienal, (E)-2-tridecenal, (2E)-2-dodecenal, (2E,6Z)-2,6-nonadienal, (E,E)-2,6-nonadienal, (2E)-2,4-undecadienal, (2E)-2,4-dodecadienal, (E)-2-nonenal, (2E,4E,7Z)-decatrienal, (Z)-2-decenal, (E)-2-octenal, (E)-2-decenal, (Z)-4-(benzyloxy)but-2-enal , (E)-4-(benzyloxy)but-2-enal, (E)-4-cyclohexylidenebut-2-enal, (2E)-3-(4-methylphenyl)-2-propenal, (2E,5E)-6,10-dimethyl-2,5,9-undecatrienal, (2E)-7,8-dimethyl-2,7-nonadienylcyclohexyl, (5E)-7-oxo-5-heptenoic acid methyl ester, (2E)-7-methyl-2,6-octadienal, (2E)-6,6-dimethyl-2-heptenal, (+-)-(2E)-5,9-dimethyl-2,8-decadienal, (E)-2-tetradecenal, and (E)-8-methoxy-4,8-dimethylnon-2-enal.

[0191] In one embodiment, the receptor is OR10J5, the agonist odorant compound comprises a lily of the valley compound; and the selected compound comprises at least one positive allosteric modulator selected from the group consisting of: octanal; (E)-dec-2-enal, 2-phenylpropanal; (E)-but-2-enal; 3-methylbenzaldehyde; 3-(1,3-benzodioxazol-5-yl)-2-methylpropanal; (+-)-3-(4-methyl-3-penten-1-yl)-3-cyclohexene-1-carboxaldehyde, (+-)-4-(4-methyl-3-penten-1-yl)-3-cyclohexene-1-carboxaldehyde, and mixtures thereof; heptaldehyde, 4-propan-2-ylbenzaldehyde; ( 3R)-3,7-dimethyloct-6-enal; (+)-(3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal, (+)-(3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]butanal, and mixtures thereof; hexanal; 2,6-dimethylhept-5-enal; benzaldehyde; 2-methyl-3-(4-methylphenyl)propanal; 3,5,6-trimethyl-3-cyclohexene-1-carboxaldehyde, 2,4,6-trimethyl-3-cyclohexene-1-carboxaldehyde, and mixtures thereof; 4-ethylbenzaldehyde; 6-methoxy-2,6-dimethylheptanal; (E)-non-2-enal; and combinations thereof.

[0192] In one form, positive allosteric modulators specifically enhance the activity of a particular receptor independently of the receptor agonist (activating compound).

[0193] Methods for monitoring OR activity

[0194] As long as the function of OR is not impaired, it can be used in any form in the methods or assays described herein. For example, OR can be used as follows: tissues or cells that inherently express OR, such as olfactory neurons isolated from living organisms and their culture products; olfactory cell membranes with OR; recombinant cells genetically modified to express OR and their culture products; membranes of recombinant cells; and artificial lipid bilayer membranes with OR.

[0195] In another embodiment, the indicator for monitoring olfactory receptor activity is selected from fluorescent calcium indicator dyes, calcium indicator proteins (e.g., GcaMP, a genetically encoded calcium indicator), fluorescent cAMP indicators, cell flow assays, cell dynamic mass redistribution assays, label-free cell assays, cAMP response element (CRE)-mediated reporter proteins, biochemical cAMP HTRF assays, beta-arrestin assays, or electrophysiological recordings. In particular, calcium indicator dyes are selected that can be used to monitor the activity of olfactory receptors expressed on the membrane of olfactory neurons (e.g., Fura-2AM).

[0196] In a specific embodiment, compounds are screened sequentially using fluorescence microscopy or fluorescence activated cell sorting (FACS) and odor-dependent changes in calcium dye fluorescence are measured.

[0197] In another embodiment, a molecular 3D receptor model of an olfactory receptor is used to assess binding potential in silico and identify compounds that can activate, mimic, block, inhibit, modulate and / or enhance the activity of an olfactory receptor.

[0198] For example, a glass microelectrode or FACS machine connected to a micromanipulator is used to isolate olfactory neurons activated by a target agonist, such as a musk or floral compound. Mouse olfactory sensory neurons are screened by Ca2+ imaging, similar to previously described procedures [Malnic, B., et al. Cell 96, 713–723 (1999); Araneda, RC et al. J. Physiol. 555, 743-756 (2004); and WO2014 / 210585, the entire contents of which are incorporated herein by reference. In particular, an electrically movable microscope stage is used to increase the number of cells that can be screened per experiment to at least 1,500 cells. Since there are approximately 1,200 different olfactory receptors in mice, and each olfactory sensory neuron expresses only 1 of the 1,200 olfactory receptor genes, this screening capability covers almost the entire mouse odorant receptor library. In other words, the combination of calcium imaging for high-throughput olfactory sensory neuron screening can identify nearly all odorant receptors that respond to a particular odorant. In a particular configuration, an odorant receptor that responds to a target agonist, such as a musk or floral compound, can be isolated. For example, at least one neuron is isolated.

[0199] For calcium imaging of olfactory neurons, primary olfactory epithelial cells can be dissected from mice prior to neuronal dissociation. The dissected olfactory epithelial cells can then be transferred to a dissociation buffer for mechanical and enzymatic dissociation. The dissociated neurons can then be plated onto coverslips, allowing thousands of cells to be screened by fluorescence microscopy, and the cells can be loaded with a calcium sensitive dye (Fura-2AM), for example, for about 30 minutes at 31°C and transferred to a microscope ready for screening. The cells are stimulated by perfusing a diluted odorant solution (in saline) over the dissociated olfactory neurons. The receptors are stimulated, for example, with 50 μM of a malodorous compound, and then the intracellular Ca is monitored by monitoring changes in Fura-2 fluorescence. 2+ The method is used to identify rare cells that respond to malodorous compounds by high throughput. After analysis, the responding cells can be retrieved from the glass coverslip using an aspiration micropipette. The separated cells can then be combined into one sample or processed individually to subsequently identify the odorant receptor genes expressed as mRNA in the responding cells.

[0200] In a specific embodiment, the mRNA of olfactory neurons is purified and amplified according to the methods generally described in Marko, NF, et al., (2005) A robust method for the amplification of RNA in the sense orientation. BMC genomics, 6, 27; doi: 10.1186 / 1471-2164-6-27 (Eberwine method). At least a portion of the transcriptome (up to and including the entire transcriptome) is sequenced using next generation sequencing (NGS) technology or hybridized to known genes using microarray technology. NGS is generally discussed and described in Metzker, ML Nat. Rev. Genet. 11, 31–46 (2010). In a specific embodiment, at least 5 neurons that exhibit the same response profile are pooled. Immediately after selection, mRNA is released by cell lysis; no DNase and no purification step are performed. mRNA is amplified by two consecutive rounds of in vitro transcription (IVT). Amplification can be performed according to the MesageAmpII aRNA kit (Ambion, AMA1751) with the following parameters: two consecutive rounds of 14-hour long IVT.

[0201] In another embodiment, a LD-PCR (long distance polymerase chain reaction) based method is used, such as the method described in an NGS-ready kit for next generation sequencing (NGS) (e.g., Clontech / Takara, Low Input RNA Kit for Sequencing-v3, catalog number 634848) was used to purify and amplify mRNA from single olfactory neurons. Single-cell mRNA was first reverse transcribed into corresponding cDNA, then amplified using 18 PCR cycles and used as NGS samples for transcriptome sequencing.

[0202] In yet another embodiment, the identity of the group or gene family of the target agonist (e.g., musk or floral compound) olfactory receptor is determined by comparing the results of NGS readings obtained from isolated activated olfactory sensory neurons with the reference genome sequence of the same species (e.g., up to the number of neurons selected). In particular, the putative target agonist (e.g., musk or floral compound) receptor will be the most highly abundant olfactory receptor mRNA in the NGS sample derived from the olfactory neuron, or will be present in more than one independent biological replicate. Due to the combinatorial nature of the olfactory code (one compound activates many ORs, and one OR can be activated by many compounds), pooling several neurons activated by a given compound allows retrieval of almost all receptors responsible for sensing these molecules in a single NGS experiment. Therefore, pooling neurons with similar functions greatly improves the throughput and speed of the deorphanization method.

[0203] Standard bioinformatics tools are then used to identify human odorant receptors that are most closely related to other putative mammalian (non-human) target agonist (e.g., musk or floral compound) receptors, assuming that homologous sequence receptors retain similar functions. Several methods have successfully identified human OR-ligand pairs based on this assumption [Armelin-Correa and Malnic (2017)], and up to 80% of mouse-human orthologs appear to retain similar functional response profiles [Adipietro, KA, et al. PLoS Genet. 8, e1002821–e1002821 (2012)]. The default parameters of the BLASTP and / or BLASTN algorithms, or other ortholog pair identification algorithms such as InParanoid, can be used.

[0204] Target agonist (e.g., musk or floral compounds) receptors of humans or non-human mammals can be suitable for functional assays, which can be used to identify compounds that bind, inhibit, block, inhibit and / or modulate the activity of olfactory receptors. In particular, the assay can be a cell-based assay or a binding assay, and the method for identifying the compound can be a high-throughput screening assay. More specifically, provided herein is a receptor-based assay suitable for high-throughput screening of receptors with a compound library to find positive allosteric modulator compounds for a specific agonist.

[0205] In a specific embodiment, the receptor gene sequence of the target agonist (e.g., musk or floral compounds) is identified from cells that are sensitive to the target agonist as follows: the pooled neurons are heated to 75°C for 10 minutes to disrupt the cell membrane and make their mRNA available for amplification. This amplification step is important when NGS technology is applied with limited amounts of starting material (usually 1 to 15 cells). Linear amplification according to the Eberwine method (IVT) ensures that the relative transcription levels of the expressed genes are maintained. Two consecutive rounds of overnight (14h) in vitro transcription are used to produce sufficient amounts of cRNA; the amplified cRNA is then used to generate an Illumina HiSeq cDNA library. The resulting short sequences, usually 75 to 150 base pairs (usually referred to as "reads"), are aligned to the reference genome of the mouse (e.g., UCSC version mm9 or mm10) to construct the complete transcriptome of these cells. Quantitative analysis of the transcriptome data produces a list of transcribed odorant receptor genes and their respective expression levels. Odorant receptor genes showing the most abundant mRNA levels (most abundant "reads") or present in more than one replicate experiment were considered putative target agonist receptors.

[0206] The latest versions of the mouse and human genome databases are then mined using the predicted mouse OR genes to identify the most closely related receptors (i.e., highest sequence similarity) in mice (paralogous genes) and humans (orthologous genes). This process can use the BLAST search algorithm (publicly available on the NCBI website), a sequence similarity search tool in which each putative gene sequence previously obtained from the initial transcriptome analysis is used as a query sequence. Under the assumption that paralogous and orthologous genes are very likely to have similar activities, the newly identified genes identified from this data mining process are considered to be potential musk or floral scent receptors. In a specific embodiment, paired comparisons of sequence homology are performed to identify closely related receptors in mice and humans, and receptors are identified as described in WO2014 / 210585. Other methods, such as RT-PCR and microarrays or mass spectrometry, may also be used.

[0207] In another embodiment, to complete the deorphanization method, the candidate OR gene is further expressed in vitro to confirm the activity of the compound against the target compound used to isolate the olfactory sensory neurons and other structurally related compounds. The mouse receptor identified from the isolated olfactory neurons that respond to the target agonist (e.g., musk or floral fragrance compound) is tagged at its N-terminus with a short polypeptide sequence (e.g., (SEQ ID NO: 10), Rho (SEQ ID NO: 12; the first 20 amino acids of the bovine rhodopsin receptor) and / or Lucy (SEQ ID NO: 14; a cleavable leucine signal peptide sequence) tag), transiently expressed in HEK 293T cells, and stimulated with target agonists (e.g., musk or floral compounds) to confirm their identity as bona fide specific target agonist receptors. In another embodiment, the RTP1 gene can also be expressed in the cell line, either by activation of the endogenous RTP1 gene (as described in WO2016201153 A1) or by transformation or viral transduction. In this cell-based assay, the human Gα subunit Gα olf Coexpression of activates the Gs transduction pathway, which results in an increase in internal cAMP upon binding of an appropriate ligand. Alternatively, in cell-based assays, the human Gα subunit Gα 15 Coexpression of α-gamma-glutamyl-1 (Gq) activated the Gq transduction pathway, which leads to the internal Ca 2+ upon binding of the appropriate ligand. 2+ The above process and the results obtained to date were used to validate a procedure for rapid and reliable identification of target agonists for mammalian odorant receptors.

[0208] Further provided are assays for identifying compounds that are positive allosteric modulators of target agonists based on the use of specific target agonist-receptor pairs. In another embodiment, provided herein are positive allosteric modulator compounds that bind to a site on the olfactory receptor that is different from the site that the agonist binds to and enhances the activity of at least one olfactory receptor selected from OR5AN1, OR10J5, Olfr1440 or Olfr16 and are identified by the methods described herein, such as the compounds described below.

[0209] In one embodiment, the activity of a compound is determined by comparing the binding of the compound to the target agonist to the binding of the target agonist alone and to the binding of the compound alone.

[0210] In another embodiment, the compound is contacted with a receptor in combination with a target agonist, or a chimera or fragment thereof, wherein the receptor, or a chimera or fragment thereof, is expressed in a cell recombinantly modified to express the receptor polypeptide.

[0211] The activity of compounds can be determined using in vivo, ex vivo, in vitro, and synthetic screening systems.

[0212] In another embodiment, contacting is performed with liposomes or virus-induced budding membranes containing a polypeptide described herein.

[0213] In another embodiment, methods for identifying compounds that bind, inhibit, block, limit and / or modulate the activity of an olfactory receptor specific for a target agonist can be performed on whole cells or membrane fractions from cells expressing a polypeptide described herein.

[0214] In one embodiment, the ORs described herein are involved in the perception of specific target agonists (e.g., musk or lily of the valley compounds) and constitute valuable candidate receptors for identifying positive allosteric modulator compounds that, when used in combination with a target agonist, can enhance the perception of a specific target agonist.

[0215] Polypeptides suitable for use according to the invention

[0216] The present invention relates to "functional equivalents" or "analogs" or "functional mutations" of the polypeptides specifically described herein.

[0217] For example, a "functional equivalent" refers to a polypeptide which exhibits at least 1% to 10%, or at least 20%, or at least 50%, or at least 75% or at least 90% higher or lower OR activity as defined herein in a test for OR activity.

[0218] According to the invention, "functional equivalents" also encompass specific mutants which have an amino acid different from the specifically stated amino acid in at least one sequence position of the amino acid sequences described herein, but which nevertheless have one of the above-mentioned biological activities. Thus, "functional equivalents" include mutants obtainable by additions, substitutions, in particular conservative substitutions, deletions and / or inversions of one or more, for example 1 to 20, 1 to 15 or 5 to 10 amino acids, wherein the changes may occur at any sequence position, as long as they result in a mutant having the profile of the properties of the invention. Functional equivalence is also provided in particular if the activity pattern qualitatively overlaps between the mutant and the unchanged polypeptide, i.e. if, for example, an interaction with the same agonist or antagonist is observed, but at a different rate (i.e., by EC 50 or IC 50 The following table shows examples of suitable (conservative) amino acid substitutions:

[0219]

[0220] "Functional equivalents" in the above sense are also "precursors" of the polypeptides, as well as "functional derivatives" and "salts" of the polypeptides.

[0221] In this context, a "precursor" is a natural or synthetic precursor of a polypeptide that may or may not have the desired biological activity.

[0222] The expression "salts" refers to salts of carboxyl groups and acid addition salts of amino groups of the protein molecules according to the invention. Salts of carboxyl groups can be produced in a known manner and include inorganic salts, such as sodium, calcium, ammonium, iron and zinc salts, and salts formed with organic bases such as amines, such as triethanolamine, arginine, lysine, piperidine, etc. Acid addition salts, such as salts formed with inorganic acids such as hydrochloric acid or sulfuric acid, and salts formed with organic acids such as acetic acid and oxalic acid, are also encompassed by the present invention.

[0223] The "functional derivatives" of the polypeptides according to the present invention can also be produced at the functional amino acid side groups or their N-termini or C-termini using known techniques. Such derivatives include, for example: aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, which can be obtained by reacting with ammonia or with primary or secondary amines; N-acyl derivatives of free amino groups, which are generated by reacting with acyl groups; or O-acyl derivatives of free hydroxyl groups, which are generated by reacting with acyl groups.

[0224] "Functional equivalents" naturally also include polypeptides obtainable from other organisms and naturally occurring variants. For example, the area of ​​homologous sequence regions can be determined by sequence comparison, and equivalent polypeptides can be determined based on the specific parameters of the present invention.

[0225] "Functional equivalents" also include fragments, preferably single domains or sequence motifs, of a polypeptide according to the invention which, for example, exhibit the desired biological function.

[0226] Furthermore, a "functional equivalent" is a fusion protein having one of the polypeptide sequences described herein or a functional equivalent derived therefrom and at least one further functionally different heterologous sequence in functional N-terminal or C-terminal association (i.e., without substantial mutual functional impairment of the fusion protein parts). Non-limiting examples of these heterologous sequences are, for example, signal peptides, histidine anchors or enzymes.

[0227] Also included according to the invention are "functional equivalents" homologous to the specifically disclosed polypeptides. They have at least 60%, preferably at least 75%, in particular at least 80 or 85%, for example 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homology (or identity) with the specifically disclosed amino acid sequences, as calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85 (8), 1988, 2444-2448. The homology or identity expressed as a percentage of a homologous polypeptide according to the invention refers in particular to the identity expressed as a percentage of amino acid residues based on the total length of one of the amino acid sequences specifically described herein.

[0228] Identity data expressed as a percentage can also be determined with the aid of a BLAST alignment, the algorithm blastp (protein-protein BLAST) or by applying the Clustal settings as described in detail herein below.

[0229] In case of possible protein glycosylation, "functional equivalents" according to the invention include the polypeptides described herein in deglycosylated or glycosylated form, as well as modified forms of the polypeptides which can be obtained by altering the glycosylation pattern.

[0230] Such functional equivalents or homologues of the polypeptides according to the invention can be produced by mutagenesis, for example by point mutations, lengthening or shortening the protein or as described in more detail below.

[0231] Such functional equivalents or homologues of polypeptides according to the present invention can be identified by screening a combined database of mutants such as shortened mutants. For example, a diversity database of protein variants can be produced by combined mutagenesis at the nucleic acid level, such as by enzymatic connection of a synthetic oligonucleotide mixture. There are many methods for generating a database of potential homologues from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and then the synthetic gene can be connected to a suitable expression vector. The use of a degenerate genome makes it possible to provide all sequences in a mixture, which encodes a potential protein sequence set required. The synthesis method of degenerate oligonucleotides is known to those skilled in the art (e.g. Narang, SA (1983); Itakura et al. (1984) (a); Itakura et al., (1984) (b); Ike et al. (1983)). The method also encompasses the production of a polypeptide having a codon-optimized nucleotide sequence, i.e., the codon usage frequency of the host cell.

[0232] In the prior art, several techniques are known for screening gene products of combinatorial databases produced by point mutation or shortening, and for screening cDNA libraries of gene products with selected properties. These techniques can be applied to rapidly screen gene libraries produced by combinatorial mutagenesis of homologues according to the present invention. The most commonly used techniques for screening large gene libraries based on high throughput analysis include cloning gene libraries in reproducible expression vectors, transforming suitable cells with the resulting vector database, and expressing combinatorial genes under specific conditions, under which the detection of the desired activity promotes the separation of the vector encoding the gene (whose product is detected). Recursive integration mutagenesis (REM) is a technique that increases the frequency of functional mutants in a database, which can be used in conjunction with screening tests to identify homologues (Arkin and Yourvan (1992); Delgrave et al. (1993)).

[0233] Encoding nucleic acid sequences suitable for use according to the present invention

[0234] The present invention also relates to nucleic acid sequences encoding the polypeptides defined herein.

[0235] The invention also relates to nucleic acids having a certain degree of "identity" to the sequences specifically disclosed herein. "Identity" between two nucleic acids refers in each case to the identity of the nucleotides over the entire length of the nucleic acid.

[0236] For example, the identity can be calculated by the Vector NTI Suite 7.1 program of Informax Corporation (USA) using the Clustal method (Higgins DG, Sharp PM. ((1989))) with the following settings:

[0237] Multiple alignment parameters:

[0238]

[0239] Pairwise comparison parameters:

[0240]

[0241]

[0242] Alternatively, identity can be determined according to the method of Chenna et al. (2003), web page: http: / / www.ebi.ac.uk / Tools / clustalw / index.html# and the following settings:

[0243]

[0244] All nucleic acid sequences mentioned herein (single-stranded and double-stranded DNA and RNA sequences, such as cDNA and mRNA) can be produced from nucleotide building blocks by chemical synthesis in a known manner, for example, by condensation of fragments of complementary nucleic acid building blocks of each overlap of a double helix. The chemical synthesis of oligonucleotides can be carried out in a known manner, for example, by the phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides, and the filling of vacancies by means of Klenow fragments of DNA polymerase and ligation reactions, and general cloning techniques are described in Sambrook et al. (1989), see below.

[0245] The invention also relates to nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, such as cDNA and mRNA) encoding one of the above-mentioned polypeptides and functional equivalents thereof, which can be obtained, for example, using artificial nucleotide analogs.

[0246] The present invention relates to isolated nucleic acid molecules encoding a polypeptide according to the invention or a biologically active segment thereof, and to nucleic acid fragments which can be used, for example, as hybridization probes or primers for identifying or amplifying encoding nucleic acids according to the invention.

[0247] The nucleic acid molecules according to the invention may additionally comprise untranslated sequences from the 3' and / or 5' end of the coding genetic region.

[0248] The invention further relates to nucleic acid molecules which are complementary to the specifically described nucleotide sequences or to segments thereof.

[0249] The nucleotide sequences according to the invention make it possible to generate probes and primers that can be used to identify and / or clone homologous sequences in other cell types and organisms. Such probes or primers typically contain a nucleotide sequence region that hybridizes under "stringent" conditions (see below) to at least about 12, preferably at least about 25, for example about 40, 50 or 75 consecutive nucleotides of the sense strand or corresponding antisense strand of the nucleic acid sequence according to the invention.

[0250] An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid, and may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or free of chemical precursors or other chemicals if chemically synthesized.

[0251] Can separate nucleic acid molecules according to the present invention by means of standard techniques of molecular biology and sequence information provided according to the present invention.For example, can use one of concrete disclosed complete sequence or its fragment as hybridization probe and standard hybridization technique (for example, be described in Sambrook, (1989)) separate cDNA from suitable cDNA library.In addition, the nucleic acid molecules that comprises one of disclosed sequence or its fragment can use oligonucleotide primer based on this sequence construction, separate by polymerase chain reaction.The nucleic acid increased in this way can be cloned in the suitable vector, and can be characterized by DNA sequencing.Oligonucleotide according to the present invention also can be prepared by standard synthetic method, for example, using automatic DNA synthesizer.

[0252] According to nucleic acid sequences of the present invention or derivatives thereof, homologues or parts of these sequences can be separated from other bacteria, for example, by conventional hybridization techniques or PCR techniques, for example by genome or cDNA libraries. These DNA sequences hybridize with sequences according to the present invention under standard conditions.

[0253] "Hybridization" refers to the ability of a polynucleotide or oligonucleotide to bind to nearly complementary sequences under standard conditions, without nonspecific binding between non-complementary pairs occurring under these conditions. For this purpose, the sequences may be 90-100% complementary. The property of complementary sequences to be able to bind specifically to each other is used, for example, in Northern or Southern blotting or in primer binding in PCR or RT-PCR.

[0254] Short oligonucleotides in conserved regions are advantageously used for hybridization. However, longer nucleic acid fragments of the invention or complete sequences may also be used for hybridization. These standard conditions vary depending on the nucleic acid used (oligonucleotides, longer fragments or complete sequences) or the type of nucleic acid used for hybridization (DNA or RNA). For example, the melting temperature of DNA:DNA hybrids is about 10°C lower than that of DNA:RNA hybrids of the same length.

[0255] For example, depending on the specific nucleic acid, standard conditions refer to a temperature of 42 to 58° C. in a buffered aqueous solution with a concentration of 0.1 to 5×SSC (1×SSC=0.15M NaCl, 15mM sodium citrate, pH 7.2), or additionally in the presence of 50% formamide (e.g., 42° C., 5×SSC, 50% formamide). Advantageously, the hybridization conditions for DNA:DNA hybrids are 0.1×SSC at a temperature of about 20° C. to 45° C., preferably about 30° C. to 45° C. For DNA:RNA hybrids, the hybridization conditions are advantageously 0.1×SSC and a temperature of about 30° C. to 55° C., preferably about 45° C. to 55° C. These stated hybridization temperatures are examples of calculated melting temperature values ​​for a nucleic acid of about 100 nucleotides in length and a G+C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization have been described in relevant genetics textbooks (e.g., Sambrook et al., 1989) and can be calculated using formulas known to those skilled in the art, for example depending on the length of the nucleic acid, the type of hybrid or the G+C content. Those skilled in the art can obtain more information about hybridization from the following textbooks: Ausubel et al. (eds), (1985), Brown (ed) (1991).

[0256] "Hybridization" can be performed in particular under stringent conditions. Such hybridization conditions are described, for example, in Sambrook (1989), or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

[0257] As used herein, the term "hybridizes or hybridizes under certain conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions can be such that sequences of at least about 70%, such as at least about 80%, and such as at least about 85%, 90%, or 95% identity remain bound to each other. Definitions of low stringency, medium, and high stringency hybridization conditions are provided herein.

[0258] Those skilled in the art can select appropriate hybridization conditions with minimal experimentation as illustrated by, for example, Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). In addition, stringent conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0259] As used herein, the defined low stringency conditions are as follows. The filter containing DNA is pretreated at 40°C for 6 hours in a solution containing 35% formamide, 5xSSC, 50mM Tris-HCl (pH 7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and 5-20x10 6 32P-labeled probe. Incubate the filter in the hybridization mixture at 40°C for 18-20 hours, then wash at 55°C for 1.5 hours. In a solution containing 2x SSC, 25mM Tris-HCl (pH 7.4), 5mM EDTA and 0.1% SDS. Replace the wash solution with fresh solution and incubate at 60°C for another 1.5 hours. Blot the filter dry and perform autoradiography.

[0260] As used herein, the defined medium stringency conditions are as follows. The filter containing DNA is pretreated at 50°C for 7 hours in a solution containing 35% formamide, 5x SSC, 50mM Tris-HCl (pH 7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and 5-20x10 6 32P-labeled probe. The filter was incubated in hybridization mixture at 50°C for 30 hours and then washed at 55°C for 1.5 hours. In a solution containing 2x SSC, 25mM Tris-HCl (pH 7.4), 5mM EDTA and 0.1% SDS. The wash solution was replaced with fresh solution and incubated at 60°C for another 1.5 hours. The filter was blotted dry and subjected to autoradiography.

[0261] As used herein, the defined high stringency conditions are as follows. The DNA-containing filter is prehybridized at 65°C for 8 hours to overnight in a buffer consisting of 6x SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500 μg / ml denatured salmon sperm DNA. 6 The filters were hybridized at 65°C for 48 hours in a prehybridization mixture containing cpm 32P-labeled probe. The filters were washed at 37°C for 1 hour in a solution containing 2xSSC, 0.01% PVP, 0.01% Ficoll and 0.01% BSA. They were then washed in 0.1x SSC at 50°C for 45 minutes.

[0262] If the above conditions are not suitable (eg, as for interspecies hybridization), other low, medium, and high stringency conditions well known in the art (eg, for interspecies hybridization) can be used.

[0263] The invention also relates to derivatives of the specifically disclosed or derivable nucleic acid sequences.

[0264] Thus, further nucleic acid sequences according to the invention may be derived from the sequences specifically disclosed herein and may differ therefrom by addition, substitution, insertion or deletion of single or several nucleotides and also encode polypeptides having the desired profile of properties.

[0265] The invention also encompasses nucleic acid sequences which contain so-called silent mutations or which, depending on the codon usage of the particular origin or host organism, have been altered compared to the specifically described sequence, as well as naturally occurring variants thereof, such as splice variants or allelic variants.

[0266] Also contemplated are sequences obtainable by conservative nucleotide substitutions, ie, the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility.

[0267] The present invention also relates to molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. These genetic polymorphisms may exist between individuals in a population due to natural variation. These natural variations typically produce 1-5% variation in the nucleotide sequence of a gene.

[0268] Derivatives of the nucleic acid sequences according to the invention are, for example, allelic variants which have at least 60% homology, preferably at least 80% homology, very particularly preferably at least 90% homology over the entire amino acid range at the derived amino acid level (with regard to homology at the amino acid level, reference should be made to the detailed information given above for polypeptides). Advantageously, the homology may be higher over partial regions of the sequence.

[0269] Furthermore, derivatives are also to be understood as homologues of the nucleic acid sequences according to the invention, for example homologues of animals, plants, fungi or bacteria, shortened sequences, single-stranded DNA or RNA of coding and non-coding DNA sequences. For example, at the DNA level, homologues have at least 40%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80% homology over a given entire DNA region in the sequences specifically disclosed herein.

[0270] Furthermore, derivatives are to be understood as fusions, for example, with promoters. The promoters added to the nucleotide sequence may be modified by at least one nucleotide exchange, at least one insertion, inversion and / or deletion, although without impairing the function or efficacy of the promoter. Furthermore, the efficacy of promoters may be increased by changing their sequence, or may be completely exchanged with more effective promoters or even promoters of organisms of different genera.

[0271] Generation of functional peptide mutants

[0272] In addition, the skilled person is familiar with methods for generating functional mutants, that is, nucleotide sequences encoding polypeptides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the sequences in SEQ ID NO: 2, 4, 6 or 8; and / or encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 3, 5 or 7.

[0273] Depending on the technology used, those skilled in the art can introduce completely random or more targeted mutations into genes or non-coding nucleic acid regions (e.g., important for regulating expression), and subsequently generate genetic libraries. The molecular biology methods required for this purpose are known to the skilled person and are described, for example, in Sambrook and Russell, Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001.

[0274] Methods for modifying genes and thereby modifying the polypeptides encoded thereby have long been known to those skilled in the art, for example:

[0275] - site-specific mutagenesis, in which single or multiple nucleotides of a gene are replaced in a targeted manner (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey),

[0276] - saturation mutagenesis, in which the codon for any amino acid can be exchanged or added at any site in the gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22: 1593; Barettino D, Feigenbutz M, Valcárel R, Stunnenberg HG (1994) Nucleic Acids Res 22: 541; Barik S (1995) Mol Biotechnol 3: 1),

[0277] - Error-prone polymerase chain reaction, in which the nucleotide sequence is mutated by an error-prone DNA polymerase (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18: 3739);

[0278] - SeSaM method (sequence saturation method), in which preferred exchange is prevented by the polymerase. Schenk et al., Biospektrum, Vol. 3, 2006, 277-279,

[0279] - gene propagation in mutant strains in which the mutation rate of the nucleotide sequence is increased, for example due to defects in DNA repair mechanisms (Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E. coli mutator strain. In: Trower MK (Ed.) In vitro mutagenesis protocols. Humana Press, New Jersey), or

[0280] -DNA shuffling, in which a group of closely related genes is created and digested, and these fragments are used as templates for polymerase chain reactions, in which, through repeated strand separation and recombination, the full-length mosaic gene is ultimately generated (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747).

[0281] Using so-called directed evolution (described in particular in Reetz MT and Jaeger KE (1999), Topics Curr Chem 200: 31; Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods for optimizing industrial polypeptides by directed evolution, In: Demain AL, Davies JE (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), a skilled worker can produce functional mutants on a large scale in a directed manner. To this end, in a first step, first, a gene library of the respective polypeptide is generated, for example using the methods given above. The gene library is expressed in a suitable manner, for example by bacterial or phage display systems.

[0282] The relevant gene of the host organism expressing the functional mutant (its function corresponds to the desired characteristic to a large extent) can be submitted to another mutation cycle. The step of mutation and selection or screening can be repeated iteratively until the functional mutant of the present invention has the desired characteristic to a sufficient degree. Using this iterative process, a limited number of mutations, such as 1, 2, 3, 4 or 5 mutations, can be carried out in stages, and their impact on the studied activity can be evaluated and selected. Then the selected mutant can be subjected to further mutation steps in the same manner. In this way, the number of single mutants to be studied can be significantly reduced.

[0283] The results according to the invention also provide important information about the structure and sequence of related polypeptides, which is necessary for producing other polypeptides with desired modified properties in a targeted manner. In particular, so-called "hot spots" can be defined, i.e. sequence segments that are potentially suitable for modifying properties by introducing targeted mutations.

[0284] Information can also be derived regarding positions in the amino acid sequence where mutations may occur that may have little effect on activity and which can be designated as potential "silent mutations."

[0285] Constructs for preparing polypeptides of the invention

[0286] The nucleotide sequence as described above may be part of an expression cassette. The terms expression cassette and expression construct are used synonymously. A preferred recombinant expression construct comprises a nucleotide sequence encoding a polypeptide according to the invention and under the genetic control of a regulatory nucleic acid sequence.

[0287] In the methods used according to the invention, the expression cassette may be part of an expression vector, in particular a recombinant expression vector.

[0288] According to the invention, an "expression unit" is understood to mean a nucleic acid with expression activity, which comprises a promoter as defined herein and, after functional connection with a nucleic acid or gene to be expressed, regulates expression, i.e. the transcription and translation of said nucleic acid or said gene. It is therefore also referred to in this context as a "regulatory nucleic acid sequence". In addition to the promoter, other regulatory elements, such as enhancers, may also be present.

[0289] According to the invention, an "expression cassette" or "expression construct" is understood to be an expression unit which is functionally linked to a nucleic acid to be expressed or a gene to be expressed. Thus, in contrast to an expression unit, an expression cassette contains not only nucleic acid sequences which regulate transcription and translation, but also nucleic acid sequences which are expressed as a protein as a result of transcription and translation.

[0290] In the context of the present invention, the term "expression" or "overexpression" describes the production or increase of the intracellular activity of one or more polypeptides encoded by the corresponding DNA in a microorganism. To this end, for example, the gene can be introduced into the organism, an existing gene can be replaced by another gene, the copy number of the gene can be increased, a strong promoter can be used or a gene encoding a highly active corresponding polypeptide can be used. Alternatively, these measures can be combined.

[0291] Preferably, such constructs according to the invention comprise a promoter 5' upstream and a terminator sequence 3' downstream of the respective coding sequence and optionally further customary regulatory elements, in each case operably linked to the coding sequence.

[0292] According to the invention, a "promoter", "nucleic acid with promoter activity" or "promoter sequence" is understood to mean a nucleic acid which, when functionally linked to a nucleic acid to be transcribed, regulates the transcription of said nucleic acid.

[0293] In the present context, "functional" or "operably" connected is understood as, for example, the sequential arrangement of the following substances: one of the nucleic acids with promoter activity, and the nucleic acid sequence to be transcribed and optionally other regulatory elements (for example nucleic acid sequences that ensure nucleic acid transcription) and, for example, terminators, in such a way that each regulatory element can perform its function after the nucleic acid sequence is transcribed. This does not necessarily require a direct connection in a chemical sense. Genetic control sequences, such as enhancer sequences, can even act on the target sequence from a more distant position or even from other DNA molecules. A preferred arrangement is such that the nucleic acid sequence to be transcribed is located behind the promoter sequence (i.e., the 3' end), so that the two sequences are covalently linked together. The distance between the promoter sequence and the nucleic acid sequence to be recombinantly expressed can be less than 200 base pairs, or less than 100 base pairs or less than 50 base pairs.

[0294] Besides promoters and terminators, the following may be mentioned as examples of further regulatory elements: targeting sequences, enhancers, polyadenylation signals, selection markers, amplification signals, replication origins, etc. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0295] The nucleic acid construct according to the invention comprises in particular a polypeptide encoding, for example, a polypeptide derived from SEQ ID NO. 1, 3, 5 or 7 or its reverse complementary sequence, or a derivative and homologue thereof, as well as a nucleic acid sequence which can be derived therefrom and which has been operably or functionally linked to one or more regulatory signals for advantageously controlling, for example, increasing gene expression.

[0296] In addition to these regulatory sequences, the natural regulation of these sequences may still be present before the actual structural gene and may optionally have been genetically modified so that the natural regulation has been turned off and the expression of the gene has been enhanced. However, the nucleic acid construct may also have a simpler construction, i.e., no additional regulatory signals are inserted before the coding sequence and the natural promoter with regulatory effect has not been removed. Instead, the natural regulatory sequences are mutated so that regulation no longer occurs and gene expression is increased.

[0297] Preferred nucleic acid constructs advantageously also comprise one or more already mentioned "enhancer" sequences functionally linked to the promoter, which make it possible to enhance the expression of the nucleic acid sequence. Other advantageous sequences, such as other regulatory elements or terminators, can also be inserted at the 3' end of the DNA sequence. One or more copies of nucleic acid according to the present invention can be present in the construct. In this construct, other markers, such as genes complementary to auxotrophy or antibiotic resistance, can also be optionally present to select the construct.

[0298] Examples of suitable regulatory sequences are found in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, rhaP (rhaP BAD )SP6, lambda-PR or lambda-PL promoters, which are advantageously used in Gram-negative bacteria. Other advantageous regulatory sequences are present, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. Artificial promoters can also be used for regulation.

[0299] In order to express in the host organism, the nucleic acid construct is advantageously inserted into a carrier, for example a plasmid or a phage, which makes it possible for the optimal expression of the gene in the host. Except for plasmids and phages, carriers are also understood to be all other carriers known to those skilled in the art, i.e., for example viruses, for example SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids and linear or circular DNA. These carriers can replicate autonomously or by chromosome replication in the host organism. These carriers are further developments of the present invention.

[0300] Suitable plasmids are, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113 In pB1, λgt11 or pBdCI, in pIJ101, pIJ364, pIJ702 or pIJ361 in Streptomyces, in pUB110, pC194 or pBD214 in Bacillus, in pSA77 or pAJ667 in Corynebacterium, in pALS1, pIL2 or pBB116 in fungi, in pAG-1, YEp6, YEp13 or pEMBLYe23 in yeast, or in pLGV23, pGHlac+, pBIN19, pAK2004 or pDH51 in plants. The above plasmids are a small selection of possible plasmids. Further plasmids are well known to the skilled worker and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0301] In the further development of the vector, the vector comprising nucleic acid construct of the present invention or nucleic acid of the present invention can also be advantageously introduced into the microorganism in the form of linear DNA and integrated into the genome of the host organism by heterologous or homologous recombination. This linear DNA can be composed of linearized vectors such as plasmids, or only consists of nucleic acid construct of the present invention or nucleic acid.

[0302] For optimal expression of heterologous genes in an organism, it is advantageous to modify the nucleic acid sequence to match the specific "codon usage" used in the organism. "Codon usage" can be readily determined by computer evaluation of other known genes of the organism in question.

[0303] The expression cassette according to the present invention is produced by fusing a suitable promoter to a suitable coding nucleotide sequence and terminator or polyadenylation signal. Conventional recombination and cloning techniques are used for this purpose, for example as described in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and T. J Silhavy, ML Berman and LV Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0304] For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector, which enables optimal expression of the gene in the host. Vectors are well known to the skilled person and can be found, for example, in "cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0305] The nucleic acid and amino acid sequences identified and / or used herein are listed below:

[0306] Human OR5AN1

[0307] Homo sapiens_OR5AN1 Musk receptor

[0308] SEQ ID NO:1-DNA

[0309] atgactgggggaggaaatattacagaaatcacctatttcatcctgctgggattctcagattttcccaggatcataaaagtgctcttcactatattcctggtgatctacattacatctctggcctggaacctctccctcattgttttaataaggatggattcccacctccatacacccatgtatttcttcctcagtaacctgtccttcatagatgtctgctatatcagctccacagtccccaagatgctctccaacctcttacaggaacagcaaactatcacttttgttggttgtattattcagtactttatcttttcaacgatgggactgagtgagtcttgtctcatgacagccatggcttatgatcgttatgctgccatttgtaaccccctgctctattcatccatcatgtcacccaccctctgtgtttggatggtactgggagcctacatgactggcctcactgcttctttattccaaattggtgctttgcttcaactccacttctgtgggtctaatgtcatcagacatttcttctgtgacatgccccaactgttaatcttgtcctgtactgacactttctttgtacaggtcatgactgctatattaaccatgttctttgggatagcaagtgccctagttatcatgatatcctatggctatattggcatctccatcatgaagatcacttcagctaaaggcaggtccaaggcattcaacacctgtgcttctcatctaacagctgtttccctcttctatacatcaggaatctttgtctatttgagttccagctctggaggttcttcaagctttgacagatttgcatctgttttctacactgtggtcattcccatgttaaatcccttgatttacagtttgaggaacaaagaaattaaagatgccttaaagaggttgcaaaagagaaagtgctgctgag

[0310] SEQ ID NO: 2 - Protein

[0311] MTGGGNITEITYFILLGFSDFPRIIKVLFTIFLVIYITSLAWNLSLIVLIRMDSHLHTPMYFFLSNLSFIDVCYISSTVPKMLSNLLQEQQTITFVGCIIQYFIFSTMGLSESCLMTAMAYDRYAAICNPLLYSSIMSPTLCVWMVLGAYMTGLTASLFQIGALLQLHFCGSNVIRHFFCDMPQLLILSCTDTFFVQVMTAILTMFFGIASALVIMISYGYIGISIMKITSAKGRSKAFNTCASHLTAVSLFYTSGIFVYLSSSSGGSSSFDRFASVFYTVVIPMLNPLIYSLRNKEIKDALKRLQKRKCC

[0312] Human OR10J5

[0313] Homo sapiens_OR10J5 floral fragrance lily of the valley receptor

[0314] SEQ ID NO:3 - DNA

[0315] atgaagagaaagaacttcacagaagtgtcagaattcattttcttgggattttctagctttggaaagcatcagataaccctctttgtggttttcctaactgtctacattttaactctggttgctaacatcatcattgtgactatcatctgcattgaccatcatctccacactcccatgtatttcttcctaagcatgctggctagttcagagacggtgtacacactggtcattgtgccacgaatgcttttgagcctcatttttcataaccaacctatctccttggcaggctgtgctacacaaatgttcttttttgttatcttggccactaataattgcttcctgcttactgcaatggggtatgaccgctatgtggccatctgcagacccctgagatacactgtcatcatgagcaagggactatgtgcccagctggtgtgtgggtcctttggcattggtctgactatggcagttctccatgtgacagccatgttcaatttgccgttctgtggcacagtggtagaccacttcttttgtgacatttacccagtcatgaaactttcttgcattgataccactatcaatgagataataaattatggtgtaagttcatttgtgatttttgtgcccataggcctgatatttatctcctatgtccttgtcatctcttccatccttcaaattgcctcagctgagggctggaagaagacctttgccacctgtgtctcccacctcactgtggttattgtccactgtggctgtgcctccattgcctacctcaagccgaagtcagaaagttcaatagaaaaagaccttgttctctcagtgacgtacaccatcatcactcccttgctgaaccctgttgtttacagtctgagaaacaaggaggtaaaggatgccctatgcagagttgtgggcagaaatatttcttaa

[0316] SEQ ID NO:4 - Protein

[0317] MKRKNFTEVSEFIFLGFSSFGKHQITLFVVFLTVYILTLVANI I IVTI ICIDHHLHTPMYFFLSMLASSETVYTLVIVPRMLLSLIFHNQPISLAGCATQMFFFVILATNNCFLLTAMGYDRYVAICRPLRYTVIMSKGLCAQLVCGSFGIGLTMAVLHVTAMFNLPFCGTVVDHFFCDIYPVMKLSCIDTTINEIINYGVSSFVIFVPIGLIFISYVLVISSILQIASAEGWKKTFATCVSHLTVVIVHCGCASIAYLKPKSESSIEKDLVLSVTYTIITPLLNPVVYSLRNKEVKDALCRVVGRNIS

[0318] Mouse Olfr1440

[0319] Mus musculus_Olfr1440_Musk receptor

[0320] SEQ ID NO:5 - DNA

[0321] atgcctggagggaggaatagcacagtcatcaccaagttcatccttgtgggattctcagattttccaaagctcaagctggttctctttgttatcttcctgggaagttatctctccacagtggtgtggaacttgggcctcatcatcttgattaggattgacccttacctacacacacctatgtacttcttcctcagcaatttgtcatttttagatttctgttacatttcatctacaacccctaaaatgctctcgggattcttccagaagtctaaatctatctcctttgttgggtgcaccatgcagtacttcatcttctcaagcctgggtctgtccgaatgctgccttctggcagccatggcttatgaccggtatgctgccatttgtaatcctcttctctacacagccatcatgtccccgtcactctgtgtgcacatggtggttggagcctatagtactggtctcttgggttcattgattcaactgtgtgctatacttcagctccatttctgtgggccaaatattataaaccatttcttttgtgacctgcctcagctattagttctttcctgctctgaaacctttcccctgcaagtcttgaaatttgtaatagcagtgatttttggggtggcatctgtcattgttatcctgatatcctatggttatatcattggcacaatcctgaatatcagctcagtagaaggtaggtccaaggcattcaatacctgtgcctctcacctgacagcagtcaccctcttttttggatcaggactctttgtctatatgcgccccagctccaacagttcccagggttatgacaagatggcttccgtgttctatacagtggtgattcccatgttgaatcctctgatttatagtctcaggaacaaggaaataaaagatgctcttcagagatgtaaaaataagtgcttttctcagtgccactgttag

[0322] SEQ ID NO:6 - Protein

[0323] MPGGRNSTVITKFILVGFSDFPKLKLVLFVIFLGSYLSTVVWNLGLIILIRIDPYLHTPMYFFLSNLSFLDFCYISSTTPKMLSGFFQKSKSISFVGCTMQYFIFSSLGLSECCLLAAMAYDRYAAICNPLLYTAIMSPSLCVHMVVGAYSTGLLGSLIQLCAILQLHFCGPNIINHFFCDLPQLLVLSCSETFPLQVLKFVIAVIFGVASVIVILISYGYIIGTILNISSVEGRSKAFNTCASHLTAVTLFFGSGLFVYMRPSSNSSQGYDKMASVFYTVVIPMLNPLIYSLRNKEIKDALQRCKNKCFSQCHC

[0324] Mouse Olfr16

[0325] Mus musculus_Olfr16_Flower fragrance lily of the valley receptor

[0326] SEQ ID NO:7-DNA

[0327] Atgcagagaaataacttcactgaagtgatagagttcgtcttcctgggattctccagctttggaaagcatcagataaccctctttgtggttttcctaaccatctacattttaactctggctggcaacatcattatagtgacaatcacacacatagaccaccaccttcacactcccatgtacttctttctgagcatgttggcaagctcagagactgtgtacacactggtcattgtcccacgaatgctttccagcctgattttttacaaccttcccatatccttggcaggctgcgcaacccaaatgttcttttttgtcaccttggccaccaacaactgctttctgctcacagcaatgggttatgatcgttatgtggctatttgtaatcctctgagatatacaatcatcatgagcaagggaatgtgtgccttgttggtttgtgggtctttaggcactggcctggttatggcagttcttcatgtgccagccatgttccatttgcccttttgtggcacggtggtggagcactttttctgtgacatatacccagtaatgaagctttcttgtgttgataccactgtcaatgagataatcaattatggtgtaagttcatttgtaattcttgtgcccatagggctgatatttatctcctatgtgctcattgtctcttccatccttaaaattgtgtccactgaaggccagaagaaagcctttgccacctgtgcctctcatctcactgtggtcattgtccactatggctgtgcctccattgcctacctcaaacccaaatcagaaagttcagtagaaaaagaccttcttctctctgtgacctacactatcatcactcccttgctgaaccctgttgtctacagcctcaggaacaaagaagtcaaagatgctctatgcagagctgtgggcagaaacacttcttaa

[0328] SEQ ID NO:8 - Protein

[0329] MQRNNFTEVIEFVFLGFSSFGKHQITLFVVFLTIYILTLAGNIIIVTITHIDHHLHTPMYFFLSMLASSETVYTLVIVPRMLSSLIFYNLPISLAGCATQMFFFVTLATNNCFLLTAMGYDRYVAICNPLRYTIIMSKGMCALLVCGSLGTGLVMAVLHVPAMFHLPFCGTVVEHFFCDIYPVMKLSCVDTTVNEIINYGVSSFVILVPIGLIFISYVLIVSSILKIVSTEGQKKAFATCASHLTVVIVHYGCASIAYLKPKSESSVEKDLLLSVTYTIITPLLNPVVYSLRNKEVKDALCRAVGRNTS

[0330] Label

[0331] SEQ ID NO:9 - DNA

[0332] gattacaaggacgacgacgataag

[0333] SEQ ID NO:10 - Protein

[0334] DYKDDDDK

[0335] Rho Label

[0336] SEQ ID NO:11 - DNA

[0337] atgaacgggaccgagggcccaaacttctacgtgcctttctccaacaagacgggcgtggtg

[0338] SEQ ID NO:12 - Protein

[0339] MNGTEGPNFYVPFSNKTGVV

[0340] Lucy Label

[0341] SEQ ID NO:13 - DNA

[0342] atgagaccccagatcctgctgctcctggccctgctgaccctaggcctggct

[0343] SEQ ID NO:14 - Protein

[0344] MRPQILLLLALLTLGLA

[0345] Motif

[0346] SEQ ID NO:15

[0347] MAYDRYVAIC

[0348] Motif

[0349] SEQ ID NO:16

[0350] FSTCSSH

[0351] Motif

[0352] SEQ ID NO:17

[0353] PMLNPFIY

[0354] The following examples are illustrative only and are not intended to limit the scope of the invention described in the abstract, description, or claims.

[0355] Example

[0356] Example 1: Identification of odorant receptors associated with sensory outcomes.

[0357] Select various target compounds (e.g., musk, floral or woody components) that are important to the perfume industry. Methods such as those described in WO2014 / 210585 can be used to identify a relatively small fraction of all ORs present in an organism, which are specifically activated or inhibited by one or more of these compounds. Transcriptomic analysis (e.g., NGS methods) or proteomic analysis (e.g., mass spectrometry) represent other methods for in vitro identification of ORs. Alternatively, in vivo methods (e.g., DREAM methods) allow OR deorphanization during gas phase delivery of volatile compounds [Von Der Weid B. et al., Nature neuroscience, 18 (10): 1455-63 (2015)].

[0358] Olfactory receptors can be evaluated to determine whether the psychological (sensory) outcome perceived by the target compound can be correlated with the activity induced by the target compound in vitro. Such olfactory receptors that appear to have predictive power for the putative sensory outcome are selected for further screening of receptor-based enhancement directed by the target compound. Alternatively, olfactory receptors activated by specific agonists of interest to the perfume industry are selected. Specific target receptor-agonist pairs are used in assays or methods for screening and identifying positive allosteric modulator compounds of the target agonist.

[0359] Example 2: Identification of Musk Olfactory Receptors

[0360] In terms of the average odor detection threshold at the sensory level, musk compounds muscone, Musk xylene and musk ketone formed a highly correlated group. To test whether the sensory correlations observed between the four musk compounds were related to the activity of individual odorant receptors, the responses of the OR5AN1 olfactory receptor to the musk compounds were assessed. The observed receptor specificity and sensitivity values ​​were compared with human psychological (sensory) odor detection threshold data ( Figure 1 ).

[0361] OR5AN1 olfactory receptor is expressed by muscone, Musk xylene and musk ketone specifically activated the OR5AN1 olfactory receptor. However, none of the other musk compounds tested activated the OR5AN1 olfactory receptor. Figure 1 a.

[0362] When considering the Pearson correlations of the mean or median odor detection thresholds (sensory results) obtained for musk compounds, the observed musk receptors ( Figure 1 a) and psychophysical ( Figure 1 b) The activity ranking is the same, and the musk compound has ligand potency (EC 50 value) or efficacy (maximum span).

[0363] Psychophysical measurements showed similarities to EC 50 The results showed a strong direct correlation with the efficacy of the median odor detection threshold (r = 0.87 and r = -0.91, respectively). Similar correlation values ​​were obtained with the mean odor detection threshold. Both the median and mean sensitivities obtained at the group level followed the potency and efficacy rankings obtained in vitro (Table 1).

[0364]

[0365] In other words, the receptor activation profile fully recapitulated the psychophysical data and was sufficient to explain the observed sensory correlations between musk compounds. OR5AN1 therefore represents a good target for musk-directed receptor-based enhancement screening. Identification of more sensory key target receptors will enable screening for enhancement of other fragrance tonality (e.g., musk, floral, woody, marine).

[0366] Example 3: Identification of putative enhancers of OR5AN1 activity.

[0367] Using a cell-based assay, OR5AN1 (WO2016 / 201153A1) was tested in a HEK293T cell line that has an activated endogenous RTP1 gene and expresses odorant receptor partners. The Flag-Rho-tagged receptor was coupled to the olfactory canonical G protein Golf co-transfected and exposed to a single concentration of Binary mixtures with the test compounds.

[0368] Using a large library of volatile compounds to obtain approximately EC 10 (This concentration elicits only about 10% of the full activity level of OR5AN1 itself) Binary mixtures of 100 μg / ml were prepared. An activated cell-based assay was used for the identification of initial enhancer candidates. Receptor activity induced by a single binary mixture was detected by measuring the increase in cAMP in the cytosol using a HTRF (homogeneous time-resolved fluorescence unit) based kit (CisBio, cAMP dynamic 2 kit, 62AM4PEJ).

[0369] By The stock solution was mixed with the stock solution of each test compound to a final concentration of 10 and 500 μM, creating 481 binary mixtures. The stock solution was made of the compound dissolved in pure DMSO. Each mixture was presented to a cell line expressing the OR5AN1 olfactory receptor. Figure 2 In Figure 5, mixtures are represented by dots (●) and empty wells in plate 6 are represented by triangles (▲). The final concentration of DMSO in each binary mixture was 0.17% and had no observable effect on the cells.

[0370] The resulting activation is then measured and compared to the individual (Defines the baseline of the enhanced assay) comparison. Determine the quality of the HTS process and evaluate window variability and signal reliability by calculating the Z' value for each plate. 20 hits were obtained, 5 of which were macroketones and nitromusk OR5AN1 olfactory receptor agonists (hits 1, 2, 3, 5 and 9). See Figure 2 and Table 2. These OR5AN1 olfactory receptor agonists further demonstrate that the dynamic range of the assay window is adequate (as the responses recorded are well above baseline) and thus likely sensitive enough to detect even low levels of putative enhancement.

[0371] Fifteen candidates, labeled “enhancers” in Table 2, were identified. Surprisingly, 80% of these putative enhancers (12 / 15) belonged to the same class of chemicals: unsaturated aliphatic aldehydes. Next, two parallel dose-response experiments were performed to confirm the true enhancing properties of the potential candidates. First, a dose response was performed for each candidate to determine if it was an agonist in itself. Second, the potency of the putative modulator in the presence and absence of 250 μM concentration was assessed. EC of dose-response curve 50 Changes. Increase OR5AN1 olfactory receptor Molecules that respond but do not themselves exhibit measurable intrinsic agonist activity are considered true enhancers and are selected for further study.

[0372]

[0373]

[0374] Based on this analysis, the volatile tridecene aldehyde (TDA), an aliphatic α-β monounsaturated aldehyde, was identified. This compound does not activate the OR5AN1 olfactory receptor by itself, but interacts with When combined, Compared with the above, it can produce a significant enhancement effect. See Figure 3.

[0375] Tridecene aldehyde appears to be a potent enhancer of the OR5AN1 olfactory receptor. Addition of TDA at 250 μM resulted in EC 50 The activation of TDA was significantly reduced (approximately 21-fold change) and resulted in an overall higher maximum activation level (increased span) (Figure 3a). When tested alone, TDA did not show any significant activity except for minimal binding activity above approximately 100 μM. At this concentration, binding of TDA resulted in less activation than 8% of the activation window. Therefore, tridecenal is not considered a true agonist of OR5AN1.

[0376] The effect of TDA on musk compound-induced activation of the OR5AN1 olfactory receptor was observed with muscone, musk xylene, and musk ketone. In the presence of 250 μM TDA, all agonists showed a similar leftward shift in the dose-response curve (Figure 3b-d). This suggests that the enhancement is mediated through receptor binding events and is unlikely to be mediated by OR5AN1-independent effects (such as effects on the assay cells themselves). In repeated experiments, EC 50 The reduction ranged from 13 to 28 times ( Figure 4 ). The apparent enhancement observed with the binary mixture of musk and TDA is inconsistent with competitive binding, primarily because TDA is not an agonist, nor does it act as an antagonist. Instead, this type of data indicates a non-competitive binding site for TDA.

[0377] Tables 3a and 3b below report the effects of other compounds on EC 50 and span effects.

[0378] Table 3a:

[0379]

[0380] Table 3b:

[0381]

[0382] Example 4: Tridecenal as a positive allosteric modulator (PAM) to enhance OR5AN1 olfactory receptor activity.

[0383] Functional dose response experiments were performed to reveal the allosteric nature of the interaction between tridecene aldehyde (TDA) and OR5AN1. The level of enhancement of OR5AN1 activation was assessed with different concentrations of the enhancer. Using the same cell-based assay as described above, the effect of OR5AN1 on TDA activation was assessed in the presence of a series of concentrations of TDA spanning from 0 to 1 mM. Dose response curves were obtained by applying the simplified allosteric EC50 shift effect equation (available in Prism5, v.5.02) from the ternary complex interaction model. The recorded enhancement levels did not depend linearly on the concentration of the enhancer, and the following α (synergy factor) and K were obtained from the model: B Key parameter values ​​of (equilibrium dissociation constant of TDA): α = 15.7 and K B =259 μM. α>1 indicates positive allosteric regulation. Following this approach, two potent enhancers, 2-decenal and nonenal, exhibited allosteric regulation properties similar to TDA, with the following key parameters: 2-decenal, α=37.3, K B =264μM; nonenal, α=28.8, K B =96μM.

[0384] The complete pharmacological profile of the enhancers provides a means to rank enhancers based on: 1) the potency shift they induce when used in combination with a fragrance ingredient (e.g., a musk, floral, or woody ingredient); 2) the increase in efficacy (activity level of the target receptor) compared to the efficacy observed with the compound alone; 3) the affinity of the enhancer compound for the receptor; 4) the minimum concentration necessary for enhancement to occur; and / or 5) the most effective ratio of enhancer to fragrance compound that produces the greatest enhancing performance.

[0385] In addition and as a control, mice Similar experiments were performed with the responsive odorant receptor Olfr1440, an ortholog of OR5AN1. Tridecene aldehyde did not enhance the activity of the Olfr1440 receptor. Instead, the aldehyde caused the Olfr1440 The induced activation was slightly inhibited (Figure 5). This further supports the view that the enhancement of OR5AN1 by tridecenal is receptor-mediated and receptor-specific.

[0386] Example 5: Structure-activity relationships of positive allosteric modulators of the OR5AN1 olfactory receptor.

[0387] A chemically diverse collection of 67 volatile compounds structurally related to the originally discovered aliphatic unsaturated aldehyde structure was generated for structure-activity-relationship (SAR) analysis. These 67 molecules were tested for their enhancing properties on OR5AN1 to characterize the necessary chemical requirements.

[0388] The results were obtained in the presence of 250 μM of each compound. and compare it with the dose response curve of The dose response curves were compared by EC 50 - Fold change (increase in potency) and potency span ratio (increase in potency) were used to quantify the resulting enhancement.

[0389] By + EC of the compound 50 Divide by a single Reference EC 50 Get EC 50 By + The span of the compound is divided by the individual In particular, various aliphatic α-β mono- or polyunsaturated molecules have been compared.

[0390] The α-β-monounsaturated disubstituted aliphatic aldehydes were systematically found to exhibit the most potent enhancement. Functional group substitution, saturation modification, and other substitutions appeared to reduce or eliminate the compound's potential to enhance the receptor ( Figure 6 Other modifications around the α-β monounsaturated aliphatic aldehyde, such as substitution at the α position or cyclization of the aliphatic tail, prevent the enhancement from occurring.

[0391] The resulting general chemical structure showing the chemical features required for specific OR5AN1 enhancement has been determined ( Figure 7 ). Pairwise comparisons of dose response curves obtained with α-β-monounsaturated aldehydes and derivatized saturated or functional isomers further exemplify the necessary chemical features required for OR5AN1 enhancement (Figures 8-12). Figure 8 shows the different levels of enhancement obtained with decenal, decadienal, and decanal, and the requirement for the degree of unsaturation. Figure 9 shows the different levels of enhancement obtained with dodecenal and tangerinal, and the importance of the position of unsaturation.

[0392] Figure 10 shows the different enhancement levels obtained with nonenal, nonenol and nordecenol, and no enhancement when an alcohol functional group is used instead of an aldehyde group. Figure 11 shows the different enhancement levels obtained with decenal and decenone, and no enhancement when a ketone functional group is used. And Figure 12 shows the different enhancement levels obtained with undecenal and undecenic acid, and no enhancement when an acid functional group is used.

[0393] SAR analysis was performed to identify the best enhancers and determine the chemical requirements needed for a compound to enhance a given receptor. For OR5AN1, at least 10 effective enhancers have been identified, all of which are volatile compounds suitable for fragrance creation / design. Without intending to be bound by any particular theory, when several enhancers are at hand for creation, the subsequent sensory characterization of their intrinsic odor further allows the selection of the volatile compound that best suits the downstream application in terms of the overall tonality of the application (i.e., fragrance). In other words, a set of enhancing ingredients corresponding to each target molecule, such as musk, is generated and provides perfumers with more creative tools.

[0394] Example 6: Hypofunctional alleles can be restored in the presence of enhancers.

[0395] For any given OR gene, an olfactory receptor is typically encoded by several alleles, ranging from a single allele to more than fifteen alleles. Natural allelic variation in the nucleotide sequence of a gene or the amino acid sequence of a polypeptide it encodes can be about 1% to 5%.

[0396] Five OR5AN1 alleles are known to exist in the human population. These alleles are functionally different and exhibit different activity levels, ranging from full function (alleles 1 and 2), insufficient function (alleles 3 and 4) to loss of function (allele 5) (Figure 13).

[0397] Functionally deficient OR5AN1 alleles 3 and 4 showed significantly increased levels of activity when tested in a cell-based assay in the presence and absence of the identified enhancer tridecenal. The observed EC of OR5AN1 allele 3 50 The values ​​showed a 6.0-fold shift (from 9.0 μM to 1.5 μM in the presence of 250 μM tridecenal) and an activity span ratio of 2.02 (activity increased from 2311 to 4663 relative fluorescence HTRF units in the presence of tridecenal). 50The values ​​showed a 6.25-fold change (from 10.1 μM to 1.6 μM in the presence of 250 μM tridecenal) and an activity span ratio of 2.16 (activity increased from 2328 to 5026 relative fluorescence HTRF units in the presence of tridecenal). Both potency and efficacy showed a favorable change and resulted in an activity level comparable to The presumed stronger activity was more comparable for those carrying allele 2. These data suggest that individuals carrying these alleles may be more sensitive to fragrance applications containing OR5AN1-activating musks in the presence of potentiating fragrance ingredients.

[0398] Example 7: Human sensitivity to musk compounds is increased in the presence of an enhancer.

[0399] The odor detection threshold (ODT) test was used to assess the The ODT test consists of identifying the concentration for which 50% (preferably 66%) of the panelists can determine which of the three containers in a forced-choice triangle test contains the target compound. Two tests were performed to calculate the ODT in a mixture with a perceptible background odor of chemically similar molecules, a) the enhancer tridecenal and b) the non-enhancing volatile compound nonanal. These two molecules are chemically similar, but nonanal does not exhibit the α-β-unsaturated bond necessary for the enhancement identified in Example 5.

[0400] 28 panelists performed a series of triangle tests in which the presence of 0.1% tridecenal increased In each triangle test, three samples contained 0.1% tridecenal and one sample also contained The 27 panelists performed a series of triangular tests in which the presence of 0.1% nonanal increased the In each triangle test, three samples contained 0.1% nonanal and one sample also contained In each triangle test, panelists were asked to identify one sample that was different from the other two samples. The ODT was calculated based on the concentration at which the proportion of correct responses equaled 2 / 3 (66%, the midpoint between the chance of 1 / 3 and all correct responses of 1). When musk was mixed with the reinforcer, the ODT value was lower than that of the neutral compound: 0.09% and 0.93%, respectively, indicating that the detection level of musk was different under similar experimental conditions ( Fig.14 ).

[0401] Example 8: Identification of putative enhancers of OR10J5 activity.

[0402] The human lily of the valley odorant receptor OR10J5 was screened to identify putative enhancers using the same screening methods as described in Examples 1 to 5. The receptor tagged with Lucy-Flag-Rho was stained with the olfactory canonical G protein G olf co-transfected and exposed to a single concentration of the floral lily compound and a binary mixture of the test compound.

[0403] The dose response curves showed that OR10J5 responded to the floral bell-of-the-valley compounds 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carboxaldehyde, 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carboxaldehyde or a mixture thereof (trade names: For sale, hereinafter referred to as ) is activated, which is displayed in Fig.15 Using a cell-based assay, OR10J5 activity was tested in a HEK293T cell line in which the endogenous chaperone RTP1 gene had been activated and odorant receptor chaperones were expressed according to the methods disclosed in International Patent Application Publication No. WO2016 / 201153A1. olf co-transfected and exposed to a single concentration of in binary mixtures with the test compounds.

[0404] A library of volatile compounds was used to generate the About EC 20 (This concentration only induces about 20% of the full activity level of OR10J5 itself). An activated cell-based assay was used for the identification of initial enhancer candidates. Receptor activity induced by a single binary mixture was detected by measuring the increase in cAMP in the cytosol using a HTRF (homogeneous time-resolved fluorescence unit) based kit (CisBio, cAMPdynamic 2 kit, 62AM4PEJ).

[0405] By The stock solution was mixed with the stock solution of each test compound to a final concentration of 4.6 and 300 μM, creating 800 binary mixtures. The stock solution was made of the compound dissolved in pure DMSO. Each mixture was presented to a cell line expressing the OR10J5 olfactory receptor. The final concentration of DMSO in each binary mixture was 0.1% and had no visible effect on the cells (see Fig.16 ).

[0406] The resulting activation is then measured and compared to the individual (defining the baseline of the enhancement assay) comparison. The quality of the HTS process was determined and the window variability and signal reliability were assessed by calculating the Z' value for each plate. 45 hits were obtained, of which 8 were OR10J5 olfactory receptor agonists (1st, 2nd, 5th, 6th, 10th, 15th, 25th and 34th hits), see Table 4. These OR10J5 olfactory receptor agonists further confirmed that the dynamic range of the assay window was sufficient (because the responses recorded were well above the baseline) and therefore may be sensitive enough to detect even low levels of putative enhancement.

[0407] Thirty-seven candidates, labeled as “potentiating” in Table 5, were identified and tested in two parallel dose-response experiments to confirm the true potentiating properties of potential candidates. First, a dose-response experiment was performed for each candidate to determine if it was an agonist in itself. Second, an EC 20 The same candidate dose response curve was performed at a concentration (producing 20% ​​activity on OR10J5) to assess the increase in activity level. Molecules that showed a response of 1.5 ng / mL, beyond the EC20, but did not show measurable intrinsic activity themselves were considered true enhancers and were selected for further study. Through this analysis, 20 molecules that showed enhancing properties were identified. See Table 4.

[0408]

[0409]

[0410]

[0411]

[0412] Example 9: Characterization of the enhancing specificity of OR10J5 activity.

[0413] The identified enhancers were tested on structurally similar compounds. Enhance specificity. Obtain specificity in the presence of a stable concentration of the test compound and compare it with the dose response curve of The dose response curves were compared by EC 50 - Fold change (increase in potency) and potency span ratio (increase in potency) were used to quantify the resulting enhancement.

[0414] By + EC of the compound 50 Divide by the individual reference EC 50 Get EC 50 By + The span of the compound is divided by the individual The span ratio is obtained from the reference span.

[0415] Figure 17 shows the different levels of enhancement obtained for OR10J5 using a) heliotropin and b) 3-(4-methylcyclohexyl-3-en-1-yl)butanal, but no enhancement was present using c) heliotropin and d) cyclomethylenecitronellol. The activated OR10J5 could be specifically enhanced by heliotropin and 3-(4-methylcyclohex-3-en-1-yl)butanal, but not by structurally related compounds, as shown by the corresponding fold changes: 9.2 and 6.7 for heliotropin and 3-(4-methylcyclohex-3-en-1-yl)butanal, respectively, and 1.8 and 1.3 for heliotropin and cyclomethylenecitronellol, respectively.

[0416] Figure 18 shows that for the mouse ortholog of OR10J5, Olfr16, different levels of enhancement were obtained using neohetorical and 3-(4-methylcyclohexyl-3-en-1-yl)butanal, but no enhancement was present using heliotropin and cyclomethylenecitronellol. Activated Olfr16 can be specifically enhanced by heliotropin and 3-(4-methylcyclohexyl-3-en-1-yl)butanal, but not by structurally related compounds, as shown by the corresponding fold changes: 5 for heliotropin and 3-(4-methylcyclohexyl-3-en-1-yl)butanal, and 1.5 and 1.1 for heliotropin and cyclomethylenecitronellol, respectively. These results suggest enhancement of other mammalian olfactory receptors.

[0417] Example 10: Convalescent compounds enhancers as positive allosteric modulators (PAMs) to enhance OR10J5 olfactory receptor activity.

[0418] Functional dose-response experiments were performed to reveal the allosteric nature of the interaction between neohelichrysinaldehyde, 3-(4-methylcyclohexyl-3-en-1-yl)butanal, melonal, and heptanal with OR10J5. The level of enhancement of OR10J5 activation was assessed with different concentrations of enhancers. Using the same cell-based assay as above, the effects of OR10J5 on the activation of OR10J5 were evaluated in the presence of continuous concentrations of enhancers spanning from 0 to 600 μM. Dose response curves of , see Figure 19. These curves were obtained by applying a simplified allosteric EC50 shift effect equation (available in Prism7) from a ternary complex interaction model. The recorded enhancement levels did not depend linearly on the concentration of the enhancer, and the following α (synergy factor) and K were obtained from the model for each enhancer: BKey parameter values ​​of (equilibrium dissociation constant of enhancer): neohexymol, α=6.3, K B =10 μM; 3-(4-methylcyclohex-3-en-1-yl)butyraldehyde, α=7.5, K B =15μM; melonaldehyde, α=4.1, K B =16 μM; and heptanal, α=3.2, K B =51 μM. α>1 indicates positive allosteric regulation.

[0419] Example 11: In the presence of an enhancer, a person Increased sensitivity.

[0420] The odor detection threshold (ODT) test was used to assess the The ODT test consists of identifying the concentration for which 50% (preferably 66%) of the panelists can determine which of the three containers in a forced-choice triangle test contains the target compound. Two tests were performed to calculate the ODT in a mixture plus a perceptible background odor of chemically similar molecules, a) the reinforcing agent 3-(4-methylcyclohexyl-3-en-1-yl)butanal and b) the non-enhancing volatile compound cyclomethylene citronellol. These two molecules are chemically similar.

[0421] 28 panelists performed a series of triangular tests in which the increase of Concentration. In each triangle test, three samples contained 0.1% 3-(4-methylcyclohex-3-en-1-yl)butanal and one sample also contained 28 panelists performed a series of triangle tests in which the presence of 7% cyclomethylene citronellol increased In each triangle test, three samples contained 7% cyclomethylene citronellol and one sample also contained In each triangle test, panelists were asked to identify one sample that was different from the other two samples. Fig. 20 shown.

[0422] The proportion of correct responses was adjusted to account for the possibility of guessing using Abbott's formula (Lawless, 2010), which is frequently used in the field. Linear regression of material (enhancer vs. neutral compound) and log-transformed concentration level as explanatory variables and proportion of correct responses as the response variable yielded a significant main effect of material (p = .03) and a marginally significant interaction (p = .052), indicating that as concentration increased, when When mixed with enhancers (relative to neutral compounds, see Fig.21 ), the proportion of correct responses increased at a higher rate.

[0423] Example 12: Enhancement of OR10J5 activity is independent of OR10J5 agonists.

[0424] Enhancement of OR10J5 activity was tested using an additional agonist as well as one of the test compounds and the conditions described in the above examples. A dose response curve for (+-)-2,5-dimethyl-2-indan carbinol was obtained in the presence of a stable concentration of the test compound and compared to the dose response curve of the agonist alone. As described above, the EC 50 The resulting enhancement was quantified using fold change (increase in potency) and potency span ratio (increase in potency).

[0425] Figure 22 shows the different levels of enhancement obtained with a) heliotropin, but no enhancement with b) heliotropin. OR10J5 activated by (+-)-2,5-dimethyl-2-indancarbinol can be specifically enhanced by heliotropin, but not by structurally related compounds, as shown by the corresponding fold changes: 8.6 and 2.0 for heliotropin and heliotropin, respectively.

[0426] This indicates that the test compounds specifically enhance the activity of OR10J5 independently of the nature of the agonist (activating compound). In this particular case, (+-)-2,5-dimethyl-2-indan carbinol is a partial agonist of OR10J5, but is enhanced by heliotropin, resulting in a 1.7-fold increase in efficacy in addition to an increase in potency over the dynamic range of the dose response (see Figure 22a). This further indicates that the enhancement is mediated through receptor binding events and is unlikely to be mediated by receptor-independent effects (such as on the assay cells themselves).

[0427] Publications cited throughout this article are incorporated herein by reference in their entirety. Although various aspects of the present invention have been described above with reference to examples and preferred embodiments, it should be understood that the scope of the present invention is not limited by the foregoing description, but is defined by the appended claims appropriately interpreted under the principles of patent law. Sequence Listing <110> Firmenich SA <120> Methods for identifying positive allosteric modulators of odorant receptors <130> 10590 / WO <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 937 <212> DNA <213> Homo sapiens <400> 1 atgactgggg gaggaaatat tacagaaatc acctatttca tcctgctggg attctcagat 60 tttcccagga tcataaaagt gctcttcact atattcctgg tgatctacat tacatctctg 120 gcctggaacc tctccctcat tgttttaata aggatggatt cccacctcca tacacccatg 180 tatttcttcc tcagtaacct gtccttcata gatgtctgct atatcagctc cacagtcccc 240 aagatgctct ccaacctctt acaggaacag caaactatca cttttgttgg ttgtattatt 300 cagtacttta tcttttcaac gatgggactg agtgagtctt gtctcatgac agccatggct 360 tatgatcgtt atgctgccat ttgtaacccc ctgctctatt catccatcat gtcacccacc 420 ctctgtgttt ggatggtact gggagcctac atgactggcc tcactgcttc tttattccaa 480 attggtgctt tgcttcaact ccacttctgt gggtctaatg tcatcagaca tttcttctgt 540 gacatgcccc aactgttaat cttgtcctgt actgacactt tctttgtaca ggtcatgact 600 gctatattaa ccatgttctt tgggatagca agtgccctag ttatcatgat atcctatggc 660 tatattggca tctccatcat gaagatcact tcagctaaag gcaggtccaa ggcattcaac 720 acctgtgctt ctcatctaac agctgtttcc ctcttctata catcaggaat ctttgtctat 780 ttgagttcca gctctggagg ttcttcaagc tttgacagat ttgcatctgt tttctacact 840 gtggtcattc ccatgttaaa tcccttgatt tacagtttga ggaacaaaga aattaaagat 900 gccttaaaga ggttgcaaaa gagaaagtgc tgctgag 937 <210> 2 <211> 311 <212> PRT <213> Homo sapiens <400> 2 Met Thr Gly Gly Gly Asn Ile Thr Glu Ile Thr Tyr Phe Ile Leu Leu 1 5 10 15 Gly Phe Ser Asp Phe Pro Arg Ile Ile Lys Val Leu Phe Thr Ile Phe 20 25 30 Leu Val Ile Tyr Ile Thr Ser Leu Ala Trp Asn Leu Ser Leu Ile Val 35 40 45 Leu Ile Arg Met Asp Ser His Leu His Thr Pro Met Tyr Phe Phe Leu 50 55 60 Ser Asn Leu Ser Phe Ile Asp Val Cys Tyr Ile Ser Ser Thr Val Pro 65 70 75 80 Lys Met Leu Ser Asn Leu Leu Gln Glu Gln Gln Thr Ile Thr Phe Val 85 90 95 Gly Cys Ile Ile Gln Tyr Phe Ile Phe Ser Thr Met Gly Leu Ser Glu 100 105 110 Ser Cys Leu Met Thr Ala Met Ala Tyr Asp Arg Tyr Ala Ala Ile Cys 115 120 125 Asn Pro Leu Leu Tyr Ser Ser Ile Met Ser Pro Thr Leu Cys Val Trp 130 135 140 Met Val Leu Gly Ala Tyr Met Thr Gly Leu Thr Ala Ser Leu Phe Gln 145 150 155 160 Ile Gly Ala Leu Leu Gln Leu His Phe Cys Gly Ser Asn Val Ile Arg 165 170 175 His Phe Phe Cys Asp Met Pro Gln Leu Leu Ile Leu Ser Cys Thr Asp 180 185 190 Thr Phe Phe Val Gln Val Met Thr Ala Ile Leu Thr Met Phe Phe Gly 195 200 205 Ile Ala Ser Ala Leu Val Ile Met Ile Ser Tyr Gly Tyr Ile Gly Ile 210 215 220 Ser Ile Met Lys Ile Thr Ser Ala Lys Gly Arg Ser Lys Ala Phe Asn 225 230 235 240 Thr Cys Ala Ser His Leu Thr Ala Val Ser Leu Phe Tyr Thr Ser Gly 245 250 255 Ile Phe Val Tyr Leu Ser Ser Ser Ser Gly Gly Ser Ser Ser Phe Asp 260 265 270 Arg Phe Ala Ser Val Phe Tyr Thr Val Val Ile Pro Met Leu Asn Pro 275 280 285 Leu Ile Tyr Ser Leu Arg Asn Lys Glu Ile Lys Asp Ala Leu Lys Arg 290 295 300 Leu Gln Lys Arg Lys Cys Cys 305 310 <210> 3 <211> 930 <212> DNA <213> Homo sapiens <400> 3 atgaagagaa agaacttcac agaagtgtca gaattcattt tcttgggatt ttctagcttt 60 ggaaagcatc agataaccct ctttgtggtt ttcctaactg tctacatttt aactctggtt 120 gctaacatca tcattgtgac tatcatctgc attgaccatc atctccacac tcccatgtat 180 ttcttcctaa gcatgctggc tagttcagag acggtgtaca cactggtcat tgtgccacga 240 atgcttttga gcctcatttt tcataaccaa cctatctcct tggcaggctg tgctacacaa 300 atgttctttt ttgttatctt ggccactaat aattgcttcc tgcttactgc aatggggtat 360 gaccgctatg tggccatctg cagacccctg agatacactg tcatcatgag caagggacta 420 tgtgcccagc tggtgtgtgg gtcctttggc attggtctga ctatggcagt tctccatgtg 480 acagccatgt tcaatttgcc gttctgtggc acagtggtag accacttctt ttgtgacatt 540 tacccagtca tgaaactttc ttgcattgat accactatca atgagataat aaattatggt 600 gtaagttcat ttgtgatttt tgtgcccata ggcctgatat ttatctccta tgtccttgtc 660 atctcttcca tccttcaaat tgcctcagct gagggctgga agaagacctt tgccacctgt 720 gtctcccacc tcactgtggt tattgtccac tgtggctgtg cctccattgc ctacctcaag 780 ccgaagtcag aaagttcaat agaaaaagac cttgttctct cagtgacgta caccatcatc 840 actcccttgc tgaaccctgt tgtttacagt ctgagaaaca aggaggtaaa ggatgcccta 900 tgcagagttg tgggcagaaa tatttcttaa 930 <210> 4 <211> 309 <212> PRT <213> Human (Homo sapiens) <400> 4 Met Lys Arg Lys Asn Phe Thr Glu Val Ser Glu Phe Ile Phe Leu Gly 1 5 10 15 Phe Ser Ser Phe Gly Lys His Gln Ile Thr Leu Phe Val Val Phe Leu 20 25 30 Thr Val Tyr Ile Leu Thr Leu Val Ala Asn Ile Ile Ile Val Thr Ile 35 40 45 Ile Cys Ile Asp His His Leu His Thr Pro Met Tyr Phe Phe Leu Ser 50 55 60 Met Leu Ala Ser Ser Glu Thr Val Tyr Thr Leu Val Ile Val Pro Arg 65 70 75 80 Met Leu Leu Ser Leu Ile Phe His Asn Gln Pro Ile Ser Leu Ala Gly 85 90 95 Cys Ala Thr Gln Met Phe Phe Phe Val Ile Leu Ala Thr Asn Asn Cys 100 105 110 Phe Leu Leu Thr Ala Met Gly Tyr Asp Arg Tyr Val Ala Ile Cys Arg 115 120 125 Pro Leu Arg Tyr Thr Val Ile Met Ser Lys Gly Leu Cys Ala Gln Leu 130 135 140 Val Cys Gly Ser Phe Gly Ile Gly Leu Thr Met Ala Val Leu His Val 145 150 155 160 Thr Ala Met Phe Asn Leu Pro Phe Cys Gly Thr Val Val Asp His Phe 165 170 175 Phe Cys Asp Ile Tyr Pro Val Met Lys Leu Ser Cys Ile Asp Thr Thr 180 185 190 Ile Asn Glu Ile Ile Asn Tyr Gly Val Ser Ser Phe Val Ile Phe Val 195 200 205 Pro Ile Gly Leu Ile Phe Ile Ser Tyr Val Leu Val Ile Ser Ser Ile 210 215 220 Leu Gln Ile Ala Ser Ala Glu Gly Trp Lys Lys Thr Phe Ala Thr Cys 225 230 235 240 Val Ser His Leu Thr Val Val Ile Val His Cys Gly Cys Ala Ser Ile 245 250 255 Ala Tyr Leu Lys Pro Lys Ser Glu Ser Ser Ile Glu Lys Asp Leu Val 260 265 270 Leu Ser Val Thr Tyr Thr Ile Ile Thr Pro Leu Leu Asn Pro Val Val 275 280 285 Tyr Ser Leu Arg Asn Lys Glu Val Lys Asp Ala Leu Cys Arg Val Val 290 295 300 Gly Arg Asn Ile Ser 305 <210> 5 <211> 948 <212> DNA <213> Mouse (Mus musculus) <400> 5 atgcctggag ggaggaatag cacagtcatc accaagttca tccttgtggg attctcagat 60 tttccaaagc tcaagctggt tctctttgtt atcttcctgg gaagttatct ctccacagtg 120 gtgtggaact tgggcctcat catcttgatt aggattgacc cttacctaca cacacctatg 180 tacttcttcc tcagcaattt gtcattttta gatttctgtt acatttcatc tacaacccct 240 aaaatgctct cgggattctt ccagaagtct aaatctatct cctttgttgg gtgcaccatg 300 cagtacttca tcttctcaag cctgggtctg tccgaatgct gccttctggc agccatggct 360 tatgaccggt atgctgccat ttgtaatcct cttctctaca cagccatcat gtccccgtca 420 ctctgtgtgc acatggtggt tggagcctat agtactggtc tcttgggttc attgattcaa 480 ctgtgtgcta tacttcagct ccatttctgt gggccaaata ttataaacca tttcttttgt 540 gacctgcctc agctattagt tctttcctgc tctgaaacct ttcccctgca agtcttgaaa 600 tttgtaatag cagtgatttt tggggtggca tctgtcattg ttatcctgat atcctatggt 660 tatatcattg gcacaatcct gaatatcagc tcagtagaag gtaggtccaa ggcattcaat 720 acctgtgcct ctcacctgac agcagtcacc ctcttttttg gatcaggact ctttgtctat 780 atgcgcccca gctccaacag ttcccagggt tatgacaaga tggcttccgt gttctataca 840 gtggtgattc ccatgttgaa tcctctgatt tatagtctca ggaacaagga aataaaagat 900 gctcttcaga gatgtaaaaa taagtgcttt tctcagtgcc actgttag 948 <210> 6 <211> 315 <212> PRT <213> Mouse (Mus musculus) <400> 6 Met Pro Gly Gly Arg Asn Ser Thr Val Ile Thr Lys Phe Ile Leu Val 1 5 10 15 Gly Phe Ser Asp Phe Pro Lys Leu Lys Leu Val Leu Phe Val Ile Phe 20 25 30 Leu Gly Ser Tyr Leu Ser Thr Val Val Trp Asn Leu Gly Leu Ile Ile 35 40 45 Leu Ile Arg Ile Asp Pro Tyr Leu His Thr Pro Met Tyr Phe Phe Leu 50 55 60 Ser Asn Leu Ser Phe Leu Asp Phe Cys Tyr Ile Ser Ser Thr Thr Pro 65 70 75 80 Lys Met Leu Ser Gly Phe Phe Gln Lys Ser Lys Ser Ile Ser Phe Val 85 90 95 Gly Cys Thr Met Gln Tyr Phe Ile Phe Ser Ser Leu Gly Leu Ser Glu 100 105 110 Cys Cys Leu Leu Ala Ala Met Ala Tyr Asp Arg Tyr Ala Ala Ile Cys 115 120 125 Asn Pro Leu Leu Tyr Thr Ala Ile Met Ser Pro Ser Leu Cys Val His 130 135 140 Met Val Val Gly Ala Tyr Ser Thr Gly Leu Leu Gly Ser Leu Ile Gln 145 150 155 160 Leu Cys Ala Ile Leu Gln Leu His Phe Cys Gly Pro Asn Ile Ile Asn 165 170 175 His Phe Phe Cys Asp Leu Pro Gln Leu Leu Val Leu Ser Cys Ser Glu 180 185 190 Thr Phe Pro Leu Gln Val Leu Lys Phe Val Ile Ala Val Ile Phe Gly 195 200 205 Val Ala Ser Val Ile Val Ile Leu Ile Ser Tyr Gly Tyr Ile Ile Gly 210 215 220 Thr Ile Leu Asn Ile Ser Ser Val Glu Gly Arg Ser Lys Ala Phe Asn 225 230 235 240 Thr Cys Ala Ser His Leu Thr Ala Val Thr Leu Phe Phe Gly Ser Gly 245 250 255 Leu Phe Val Tyr Met Arg Pro Ser Ser Asn Ser Ser Gln Gly Tyr Asp 260 265 270 Lys Met Ala Ser Val Phe Tyr Thr Val Val Ile Pro Met Leu Asn Pro 275 280 285 Leu Ile Tyr Ser Leu Arg Asn Lys Glu Ile Lys Asp Ala Leu Gln Arg 290 295 300 Cys Lys Asn Lys Cys Phe Ser Gln Cys His Cys 305 310 315 <210> 7 <211> 930 <212> Ms <213> mice (Mus musculus) <400> 7 atgcagagaa ataacttcac tgaagtgata gagttcgtct tcctgggatt ctccagcttt 60 ggaaagcatc agataaccct ctttgtggtt ttcctaacca tctacatttt aactctggct 120 ggcaacatca ttatagtgac aatcacacac atagaccacc accttcacac tcccatgtac 180 ttctttctga gcatgttggc aagctcagag actgtgtaca cactggtcat tgtcccacga 240 atgctttcca gcctgatttt ttacaacctt cccatatcct tggcaggctg cgcaacccaa 300 atgttctttt ttgtcacctt ggccaccaac aactgctttc tgctcacagc aatgggttat 360 gatcgttatg tggctatttg taatcctctg agatatacaa tcatcatgag caagggaatg 420 tgtgccttgt tggtttgtgg gtctttaggc actggcctgg ttatggcagt tcttcatgtg 480 ccagccatgt tccatttgcc cttttgtggc acggtggtgg agcacttttt ctgtgacata 540 tacccagtaa tgaagctttc ttgtgttgat accactgtca atgagataat caattatggt 600 gtaagttcat ttgtaattct tgtgcccata gggctgatat ttatctccta tgtgctcatt 660 gtctcttcca tccttaaaat tgtgtccact gaaggccaga agaaagcctt tgccacctgt 720 gcctctcatc tcactgtggt cattgtccac tatggctgtg cctccattgc ctacctcaaa 780 cccaaatcag aaagttcagt agaaaaagac cttcttctct ctgtgaccta cactatcatc 840 actcccttgc tgaaccctgt tgtctacagc ctcaggaaca aagaagtcaa agatgctcta 900 tgcagagctg tgggcagaaa cacttcttaa 930 <210> 8 <211> 309 <212> PRT <213> Mus musculus <400> 8 Met Gln Arg Asn Asn Phe Thr Glu Val Ile Glu Phe Val Phe Leu Gly 1 5 10 15 Phe Ser Ser Phe Gly Lys His Gln Ile Thr Leu Phe Val Val Phe Leu 20 25 30 Thr Ile Tyr Ile Leu Thr Leu Ala Gly Asn Ile Ile Ile Val Thr Ile 35 40 45 Thr His Ile Asp His His Leu His Thr Pro Met Tyr Phe Phe Leu Ser 50 55 60 Met Leu Ala Ser Ser Glu Thr Val Tyr Thr Leu Val Ile Val Pro Arg 65 70 75 80 Met Leu Ser Ser Leu Ile Phe Tyr Asn Leu Pro Ile Ser Leu Ala Gly 85 90 95 Cys Ala Thr Gln Met Phe Phe Phe Val Thr Leu Ala Thr Asn Asn Cys 100 105 110 Phe Leu Leu Thr Ala Met Gly Tyr Asp Arg Tyr Val Ala Ile Cys Asn 115 120 125 Pro Leu Arg Tyr Thr Ile Ile Met Ser Lys Gly Met Cys Ala Leu Leu 130 135 140 Val Cys Gly Ser Leu Gly Thr Gly Leu Val Met Ala Val Leu His Val 145 150 155 160 Pro Ala Met Phe His Leu Pro Phe Cys Gly Thr Val Val Glu His Phe 165 170 175 Phe Cys Asp Ile Tyr Pro Val Met Lys Leu Ser Cys Val Asp Thr Thr 180 185 190 Val Asn Glu Ile Ile Asn Tyr Gly Val Ser Ser Phe Val Ile Leu Val 195 200 205 Pro Ile Gly Leu Ile Phe Ile Ser Tyr Val Leu Ile Val Ser Ser Ile 210 215 220 Leu Lys Ile Val Ser Thr Glu Gly Gln Lys Lys Ala Phe Ala Thr Cys 225 230 235 240 Ala Ser His Leu Thr Val Val Ile Val His Tyr Gly Cys Ala Ser Ile 245 250 255 Ala Tyr Leu Lys Pro Lys Ser Glu Ser Ser Ser Val Glu Lys Asp Leu Leu 260 265 270 Leu Ser Val Thr Tyr Thr Ile Ile Thr Pro Leu Leu Asn Pro Val Val 275 280 285 Tyr Ser Leu Arg Asn Lys Glu Val Lys Asp Ala Leu Cys Arg Ala Val 290 295 300 Gly Arg Asn Thr Ser 305 <210> 9 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> FLAG tag <400> 9 gattacaagg acgacgacga taag 24 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <220> <223> FLAG tag <400> 10 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 11 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Rho Tags <400> 11 atgaacggga ccgagggccc aaacttctac gtgcctttct ccaacaagac gggcgtggtg 60 <210> 12 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Rho Tags <400> 12 Met Asn Gly Thr Glu Gly Pro Asn Phe Tyr Val Pro Phe Ser Asn Lys 1 5 10 15 Thr Gly Val Val 20 <210> 13 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Lucy Tags <400> 13 atgagacccc agatcctgct gctcctggcc ctgctgaccc taggcctggc t 51 <210> 14 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Lucy Tags <400> 14 Met Arg Pro Gln Ile Leu Leu Leu Leu Ala Leu Leu Thr Leu Gly Leu 1 5 10 15 Ala <210> 15 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 15 Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys 1 5 10 <210> 16 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 16 Phe Ser Thr Cys Ser Ser His 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 17 Pro Met Leu Asn Pro Phe Ile Tyr 1 5

Claims

1. A high throughput assay system for identifying one or more compounds from a group of compounds, wherein the one or more compounds positively modulate the activity of an olfactory receptor induced by an agonist odorant compound, comprising: a) one or more isolated cells, each cell expressing a mammalian olfactory receptor, wherein the one or more cells comprise a target olfactory receptor activated by one or more odorants, b) a first compound that binds to and activates the olfactory receptor, and c) at least one second compound that binds to the receptor non-competitively with respect to the first compound and enhances the activity of the receptor exposed to the first compound compared to the activity of the receptor exposed to the first compound in the absence of the second compound, wherein the first compound is an agonist odorant compound of the olfactory receptor, wherein the first compound and the second compound are different compounds, wherein the second compound is not an agonist of the receptor, wherein the activity of the receptor is synergistically enhanced by the combination of the first compound and the second compound, and wherein the olfactory receptor is OR5AN1, the first compound comprises a musk compound selected from 3-methylcyclopentadecan-1-one, 3-methyl-5-cyclopentadecen-1-one, musk xylene and musk ketone, and the second compound comprises at least one compound selected from the group consisting of: (E,E)-2,4-decadienal, (E)-2-undecenal, (E,E)-2,4-nonadienal, (E)-2-tridecenal, (2E)-2-dodecenal, (2E,6 Z)-2,6-nonadienal, (E,E)-2,6-nonadienal, (E)-2-nonenal, (Z)-2-decenal, (E)-2-octenal, (E)-2-decenal, (5E)-7-oxo-5-heptenoic acid methyl ester, (2E)-7-methyl-2,6-octenal, (2E)-6,6-dimethyl-2-heptenal, (+-)-(2E)-5,9-dimethyl-2,8-decadienal, (E)-2-tetradecenal, and combinations thereof, and Wherein the second compound is a positive allosteric modulator of the musk olfactory receptor.

2. The assay system of claim 1, wherein the at least one second compound is selected from a group of compounds based on structure-activity relationship (SAR) analysis.

3. The assay system of claim 1 or 2, wherein the second compound binds to a site on the olfactory receptor that is different from the site to which the first compound binds.

4. The assay system of claim 1, wherein prior to step a), the one or more cells are transformed to express a target olfactory receptor.

5. The assay system of claim 1, wherein the mammalian olfactory receptor is a mouse olfactory receptor or a human olfactory receptor.

6. The assay system of claim 1, wherein the olfactory receptor comprises a polypeptide: a) the amino acid sequence of the polypeptide is SEQ ID NO: 2 or 6; and / or b) The polypeptide is encoded by a nucleic acid molecule, the nucleotide sequence of which is SEQ ID NO: 1 or 5 or its reverse complementary sequence.

7. The assay system of claim 1, wherein the modulated activity comprises: a) modulating the binding affinity of the first compound to the orthosteric site, b) modulating the binding activation efficacy of the first compound to the orthosteric site, and / or c) Regulate G protein binding sites.

8. A method for identifying at least one positive allosteric modulator compound that enhances the activity of an olfactory receptor in the presence of an agonist of the olfactory receptor, the method comprising: a) providing one or more isolated cells, each cell expressing a mammalian olfactory receptor, b) exposing the one or more cells to a first compound capable of activating the olfactory receptor, c) exposing the one or more cells to one or more test compounds, and d) selecting the at least one second compound from the one or more test compounds when the at least one second compound has a synergistic effect on receptor activity in combination with the first compound as a positive allosteric modulator compound; wherein the first compound is an agonist odorant compound of the olfactory receptor, wherein the first compound and the second compound are different compounds, wherein the second compound is not an agonist of the receptor, and wherein the olfactory receptor is OR5AN1, the first compound comprises a musk compound selected from 3-methylcyclopentadecan-1-one, 3-methyl-5-cyclopentadecan-1-one, musk xylene and musk ketone, and the one or more test compounds comprise at least one compound selected from the group consisting of: (E,E)-2,4-decadienal, (E)-2-undecenal, (E,E)-2,4-nonadienal, (E)-2-tridecenal, (2E)-2-dodecenal, (2 E,6Z)-2,6-nonadienal, (E,E)-2,6-nonadienal, (E)-2-nonenal, (Z)-2-decenal, (E)-2-octenal, (E)-2-decenal, (5E)-7-oxo-5-heptenoic acid methyl ester, (2E)-7-methyl-2,6-octenal, (2E)-6,6-dimethyl-2-heptenal, (+-)-(2E)-5,9-dimethyl-2,8-decadienal, (E)-2-tetradecenal, and combinations thereof, and The one or more test compounds are positive allosteric modulators of musk olfactory receptors.

9. The method of claim 8, wherein activation of the olfactory receptor to the first compound is measured by measuring the response of the olfactory receptor in the presence and absence of the first compound.

10. The method of claim 8 or 9, wherein the at least one second compound is selected from one or more test compounds based on structure-activity relationship (SAR) analysis.

11. The method of claim 8, wherein the second compound binds to a site on the olfactory receptor that is different from the site to which the first compound binds.

12. The method of claim 8, wherein prior to step a), the one or more cells are transformed to express the olfactory receptor.

13. The method of claim 8, wherein the olfactory receptor is heterologous to the one or more cells.

14. The method of claim 8, wherein the cell is selected from the group consisting of HEK293, CHO, Xenopus oocytes, COS, insect, and yeast.

15. A method of identifying a compound that enhances the activity of an olfactory receptor induced by an agonist odorant compound, the method comprising: (i) screening one or more compounds in a binding assay in the presence of an agonist odorant compound; (ii) selecting one or more screened compounds that can specifically enhance the specific binding or binding effect of the agonist odorant compound to the olfactory receptor of a mammal; and (iii) identifying compounds that potentially modulate the perception of an agonist odorant compound based on the specific binding or enhancement of the binding effect of the agonist odorant compound to the olfactory receptor, wherein the olfactory receptor is OR5AN1, the agonist odorant compound comprises a musk compound selected from 3-methylcyclopentadecan-1-one, 3-methyl-5-cyclopentadecen-1-one, musk xylene and musk ketone, and the selected compound comprises at least one compound selected from the group consisting of: (E,E)-2,4-decadienal, (E)-2-undecenal, (E,E)-2,4-nonadienal, (E)-2-tridecenal, (2E)-2-dodecenal, (2 E,6Z)-2,6-nonadienal, (E,E)-2,6-nonadienal, (E)-2-nonenal, (Z)-2-decenal, (E)-2-octenal, (E)-2-decenal, (5E)-7-oxo-5-heptenoic acid methyl ester, (2E)-7-methyl-2,6-octenal, (2E)-6,6-dimethyl-2-heptenal, (+-)-(2E)-5,9-dimethyl-2,8-decadienal, (E)-2-tetradecenal, and combinations thereof, and Wherein the selected compound is a positive allosteric modulator of the musk olfactory receptor.

16. The method of claim 8 or 15, wherein the mammalian olfactory receptor is a mouse olfactory receptor or a human olfactory receptor.

17. The method of claim 8 or 15, wherein the olfactory receptor comprises a polypeptide: a) the amino acid sequence of the polypeptide is SEQ ID NO: 2 or 6; and / or b) The polypeptide is encoded by a nucleic acid molecule, the nucleotide sequence of which is SEQ ID NO: 1 or 5 or its reverse complementary sequence.

18. The method of claim 8 or 15, wherein the modulated activity comprises: a) modulating the binding affinity of the first compound to the orthosteric site, b) modulating the binding activation efficacy of the first compound to the orthosteric site, and / or c) Regulate G protein binding sites.

19. The method of claim 8 or 15, wherein the enhancement of the binding of the agonist odorant compound to the olfactory receptor comprises enhancing the potency and / or efficacy of the activity of the receptor compared to the activity of the receptor in the absence of the selected compound.

20. The method of claim 8 or 15, wherein one or more selected compounds are selected based on a structure-activity relationship (SAR) analysis specifically designed to identify putative chemical determinants of potentiation.

21. The method of claim 15, wherein the screening comprises calculating an activation window to measure EC 05-50 The synergistic effect is enhanced.

22. Use of a polypeptide activated by macroketone or nitromusk, the amino acid sequence of which is SEQ ID NO: 2 or 6, in the method according to any one of claims 8 to 21.

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