Method for identifying compositions that control laundry odors, mold odors, and / or sweat odors.

Abstract

To provide a method for identifying a compound that binds, suppresses, blocks, inhibits and / or adjusts activity of an olfactory receptor activated by a laundry stench causing substance, a mold stench causing substance and / or a perspiration stench causing substance.SOLUTION: Problems are solved by identifying a panel of a specific stench causing substance (MCS) that is characteristic of a laundry stench, a mold stench, and a perspiration stench, and a panel of olfactory receptor that is activated by such a MCS and may be used as a target for identifying a regulator that counteracts activation of these olfactory receptors by such a MCS.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority over U.S. Provisional Patent Application Serial No. 62 / 589,947, filed on 22 November 2017, European Patent Application Serial No. 18157790.9, filed on 21 February 2018, and U.S. Provisional Patent Application Serial No. 62 / 754,899, filed on 2 November 2018, the entire contents of which are incorporated herein by reference.

[0002] Technical field The technical field relates to odor substances and / or aroma compounds, and odor substances and aroma receptors and assays that may be used to identify inhibitors, modifiers or neutralizers of odor compounds such as laundry odor, mold odor and / or sweat odor, e.g., dimethyl trisulfide (DMTS), geonol, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid and 3-methyl-3-sulfanylhexanol (transpyrrole).

[0003] Background technology The sense of smell is one of the most complex and poorly understood aspects of the human sensory system. From the activation of olfactory receptors (ORs) to perception, there are many steps that require further investigation. Understanding how OR codes for individual odorants and mixtures are translated into perception could yield significant benefits in several fields. These fields include odor modifiers that block the perception of unpleasant odors, such as malodorous neutralizers; novel flavor and fragrance components that replace non-biodegradable or toxic compounds; and odor enhancers that limit reliance on compounds that are difficult to obtain from natural sources. The “olfactory code” combinatorial paradigm focuses on the observation that any single OR may be activated by multiple odorants, and conversely, most odorants can activate multiple ORs. In the mouse genome, there are approximately 1200 distinctly complete ORs. In contrast, humans have approximately 400. In both cases, the OR repertoire is activated by thousands of odorants worldwide, and the complexity of this combination allows for the breadth of perceptible smells. However, as of 2014, only 82 mouse ORs (approximately 8%) and 17 human ORs (approximately 10%) had been identified as odorants or ligands using conventional de-orphanization methods. Furthermore, the physiological relevance of most ligands for human ORs, which were essentially identified in vitro, has not been tested.

[0004] A method for rapidly and reliably identifying a relatively small subset of ORs within the entire repertoire of ORs present in organisms that are specifically activated or inhibited by one or more odor molecules is described in International Publication No. 2014 / 210585 (WO2014 / 210585). However, the need remains to identify odor molecule receptors, and more specifically, malodorous receptors activated by substances that produce specific malodors, using this method.

[0005] Odor-causing compounds, such as dimethyl trisulfide (DMTS), geonol, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, and transpyrrole, can produce unpleasant odors, for example, laundry or body odor caused by sweat or sebum, or odors arising from mold.

[0006] Accordingly, the problem solved by the present invention is to provide a method for identifying modifiers or neutralizers of mold odor, laundry odor and / or sweat odor, which bind, suppress, block, inhibit, and / or modulate the activation of one or more olfactory receptors activated by specific odor-causing substances (MCS) characterized by such types of odors. Further assays are desired to identify novel compounds or compound mixtures that bind to these receptors, or that depend on novel odor receptors or newly identified odor receptors associated with specific odor-causing substances.

[0007] summary The aforementioned problem was, surprisingly, solved by identifying a panel of MCSs characteristic of laundry odors, mold odors, or sweat odors, and a panel of olfactory receptors that can be used as targets for identifying modulators that are activated by such MCSs and that counteract the activation of these olfactory receptors by such MCSs.

[0008] As MCSs for laundry, geonol, DMTS, and 1-octen-3-ol have been identified. As olfactory receptor polypeptides activated by at least one of the above compounds, human receptors OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR4S2, mouse orthologs Olfr263, Olfr403, Olfr137, Olfr173, Olfr164, Olfr735, Olfr1193, Olfr96, and mouse receptors Olfr1487, Olfr339, Olfr93, Olfr398, Olfr120, Olfr1364, and Olfr937 have been identified.

[0009] Butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, and transpyrrole have been identified as MCSs in sweat. As olfactory receptor polypeptides activated by at least one of the above compounds, human receptors OR2W1, OR1A1, OR2J3, OR5K1, OR51E1, OR51I2, mouse orthologs Olfr263, Olfr403, Olfr558, Olfr641, Olfr137, Olfr164, and mouse receptors Olfr1126 and Olfr46 have been identified.

[0010] Geonol, DMTS, and 1-octen-3-ol have been identified as mold MCSs. As olfactory receptor polypeptides activated by at least one of the above compounds, human receptors OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR4S2, mouse orthologs Olfr263, Olfr403, Olfr735, Olfr1193, Olfr96, Olfr137, Olfr173, Olfr164, and mouse receptors Olfr1487, Olfr339, Olfr93, Olfr398, Olfr120, Olfr1364, and Olfr937 have been identified.

[0011] Furthermore, the present invention provides a method for assaying whether a compound binds to, inhibits, blocks, restricts, and / or modulates the activity of olfactory receptors activated by substances that cause laundry odors, mold odors, and / or sweat odors, and in particular a method for identifying such compounds.

[0012] Furthermore, isolated olfactory receptor polypeptides, corresponding encoding nucleic acid sequences, recombinant nucleic acids containing such encoding sequences, and corresponding expression vectors are provided.

[0013] Furthermore, a non-human host organism or host cell is provided that has been modified to express such olfactory receptor polypeptides, which are activated by MCS, a characteristic of laundry odor, mold odor, or sweat odor.

[0014] Furthermore, the use of such olfactory receptor polypeptides, which are activated by MCS and are characteristic of laundry odors, mold odors, or sweat odors, is provided for identifying odor-modulating compounds. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows an MCS and olfactory receptor according to some of the embodiments presented herein. [Figure 2] Figure 2 shows the potency of the antagonist 1-[(1S,7S)-2,3,4-trimethyltricyclo[5.2.1.0~1.5~]decyl-4-yl)ethanone oxime as an inhibitor of the butyrate olfactory receptor OR51E1 under very stringent screening conditions. The black line shows the inhibition (%) of butyrate olfactory receptor activity observed in cells treated with 81 μM butyrate (EC87) and the antagonist at the indicated concentrations [μM]. [Figure 3] Figure 3 shows an overview of the inhibition (%) of the butyrate olfactory receptor OR51E1 observed under very stringent screening conditions in cells treated with compounds according to several embodiments described herein. The structures of the tested compounds are shown on both sides of the figure. [Figure 4] Figure 4 shows the ability of compounds according to several embodiments of this disclosure to reduce the perceived intensity of geonol in human sensory panel tests. Many of these compounds show inhibition of the activity of the geonol olfactory receptor. NS: Perception (%) of remaining geonol is not statistically different compared to geonol alone at isointensity concentrations; Asterisk (*): Perception (%) of remaining geonol is statistically different compared to geonol alone at isointensity concentrations. [Figure 5]FIG. 5 shows the activation of OR11A1, Olfr96, Olfr339, Olfr1487, and Olfr1322 that is dependent on and specific to the concentration of geonol in a cell-based assay according to the assays and methods disclosed in International Publication No. 2014 / 210585 and further in the paragraph of Methods of Monitoring OR Activity. The detection method is related to the OR-dependent accumulation of cAMP in the cells as a result of OR activation by geonol. [Figure 6] FIG. 6 shows the activation of Olfr93, Olfr398, Olfr120, and Olfr1364 that is dependent on and specific to the concentration of 1-octen-3-ol in a cell-based assay according to the assays and methods disclosed in International Publication No. 2014 / 210585 and further in the paragraph of Methods of Monitoring OR Activity. The detection method is related to the OR-dependent accumulation of cAMP in the cells as a result of OR activation by 1-octen-3-ol. [Figure 7] FIG. 7 shows the activation of OR2W1, Olfr263, and Olfr46 that is dependent on and specific to the concentration of trans-2-hexenal in a cell-based assay according to the assays and methods disclosed in International Publication No. 2014 / 210585 and further in the paragraph of Methods of Monitoring OR Activity. The detection method is related to the OR-dependent accumulation of cAMP in the cells as a result of OR activation by trans-2-hexenal. [Figure 8] FIG. 8 shows the activation of OR51I2, Olfr641, OR2W1, and Olfr263 that is dependent on and specific to the concentration of 3-methyl-2-hexenoic acid in a cell-based assay according to the assays and methods disclosed in International Publication No. 2014 / 210585 and further in the paragraph of Methods of Monitoring OR Activity. The detection method is related to the OR-dependent accumulation of cAMP in the cells as a result of OR activation by 3-methyl-2-hexenoic acid. [Figure 9]Figure 9 shows concentration-dependent and specific activation of Olfr1126 in a cell-based assay according to the assays and methods disclosed in International Publication No. 2014 / 210585 and further in the paragraph of Methods of Monitoring OR Activity. The detection method is related to the OR-dependent accumulation of cAMP in said cells as a result of OR activation by 3-hydroxy-3-methyl-hexanoic acid. [Figure 10] Figure 10 shows NGS data from a single OSN responding to geonol according to the methods described in International Publication No. 2014 / 210585 and further in the section of Methods of OR activity. The left panel shows the relative transcript levels of the ORs, with Olfr96 being significantly higher than the rest. The right panel shows the presence and relative amounts of the housekeeping genes (omp, actb, cnga2, rtp1, gnal, adcy3) of the major markers for the OSNs in the same NGS sample. [Figure 11] Figure 11 shows NGS data from a single OSN responding to 1-octen-3-ol according to the methods described in International Publication No. 2014 / 210585 and further in the section of Methods of OR activity. The left panel shows the relative transcript levels of the ORs, with Olfr93 being significantly higher than the rest. The right panel shows the presence and relative amounts of the housekeeping genes (omp, actb, cnga2, rtp1, gnal, adcy3) of the major markers for the OSNs in the same NGS sample. [Figure 12] Figure 12 shows NGS data from a single OSN responding to 1-octen-3-ol according to the methods described in International Publication No. 2014 / 210585 and further in the section of Methods of OR activity. The left panel shows the relative transcript levels of the ORs, with Olfr120 being significantly higher than the rest. The right panel shows the presence and relative amounts of the housekeeping genes (omp, actb, cnga2, rtp1, gnal, adcy3) of the major markers for the OSNs in the same NGS sample. [Figure 13] Figure 13 shows NGS data from a single OSN responding to 1-octen-3-ol, according to the method described in International Publication No. 2014 / 210585 and further in the section on Methods of OR activity. The left panel shows the relative transcription levels of OR, with Olfr1364 significantly higher than the rest. The right panel shows the presence and relative levels of major marker housekeeping genes (omp, actb, cnga2, rtp1, gnal, adcy3) for OSN in the same NGS sample. [Figure 14] Figure 14 shows NGS data from a single OSN responding to 1-octen-3-ol, according to the method described in International Publication No. 2014 / 210585 and further in the section on Methods of OR activity. The left panel shows the relative transcription levels of OR, with Olfr937 significantly higher than the rest. The right panel shows the presence and relative levels of housekeeping genes (omp, actb, cnga2, rtp1, gnal, adcy3) for the major markers of OSN in the same NGS sample. [Figure 15] Figure 15 shows NGS data from a single OSN responding to transpyrrole, performed according to the method described in International Publication No. 2014 / 210585 and further in the section on Methods of OR activity. The left panel shows the relative transcription levels of OR, with Olfr263 significantly higher than the rest. The right panel shows the presence and relative levels of housekeeping genes for major markers of OSN in the same NGS sample. [Figure 16] Figure 16 shows NGS data from a single OSN responding to 3-hydroxy-3-methylhexanoic acid, performed according to the method described in International Publication No. 2014 / 210585 and further in the section on Methods of OR activity. The left panel shows the relative transcription levels of ORs, with Olfr1126 significantly higher than the rest. The right panel shows the presence and relative levels of housekeeping genes for major markers of OSNs in the same NGS sample.

[0016] Detailed description of the invention definition In the specification and accompanying claims, the use of "or" means "and / or" unless otherwise specified.

[0017] Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and not intended to be restrictive.

[0018] Furthermore, when describing various embodiments using the term “comprising,” those skilled in the art will understand that in certain examples, one embodiment may instead be described using the language “consisting essentially of” or “consisting of.”

[0019] The following terms have meanings that apply to them unless otherwise specified.

[0020] "OR" refers to one or more members of the family of G protein-coupled receptors (GPCRs) expressed in olfactory cells. Olfactory receptor cells can be identified based on morphology or by the expression of proteins specifically expressed in olfactory cells. OR family members may have the ability to act as receptors for olfactory signaling.

[0021] "DMTS OR" refers to a member of the family of G protein-coupled receptors expressed in olfactory cells, which are bound and / or activated by DMTS in binding or activation assays for identifying ligands that bind and / or activate GPCRs. Such assays are described below. DMTS receptors as used herein include variants, including fragments, synthetic and naturally occurring ones, as well as chimeric or recombinant nucleic acids or proteins that respond to or bind to DMTS. Similar definitions apply analogously to the following receptors: "Geonol OR", "1-octen-3-ol OR", "butyric acid OR", "3-methyl-2-hexenoic acid OR", "3-hydroxy-3-methylhexanoic acid OR", and "transpyrrole OR".

[0022] An "OR" polypeptide is considered to be one that belongs to a seven-transmembrane domain G protein-coupled receptor superfamily encoded by a single long exon of ~1kb, and exhibits characteristic olfactory receptor-specific amino acid motifs. The seven domains are called TM I to TM VII, which are "transmembrane" domains or "TM" domains connected by three "internal cell loop" or "IC" domains, IC I to IC III, and three "external cell loop" or "EC" domains, EC I to EC III. The motifs and their variants are defined, but are not limited to, the MAYDRYVAIC motif overlapping with TM III and IC II (SEQ ID NO: 53), the FSTCSSH motif overlapping with IC III and TM VI (SEQ ID NO: 54), the PMLNPFIY motif in TM VII (SEQ ID NO: 55), and the presence of three conserved C residues in EC II and a 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)].

[0023] The "OR" nucleic acid encodes a family of GPCRs having a seven-transmembrane domain with "G protein-coupled receptor activity," such as those that bind to G proteins in response to extracellular stimuli and, upon stimulation by enzymes such as phospholipase C and adenylyl cyclase, transmit second messengers such as IP3, cAMP, cGMP, and Ca 2+ The production of may be promoted.

[0024] A "paralogus" or "paralog" gene is a result of gene duplication and refers to a closely related homologous gene within the same species.

[0025] An "ortholog" gene is defined as a homologous gene that is phylogenetically related to another species by a gene present in a common ancestor.

[0026] The "N-terminal domain" region begins at the N-terminus and extends to a region close to the beginning of the first transmembrane domain.

[0027] The “transmembrane region” includes seven “transmembrane domains,” which are domains of the OR polypeptide located within the plasma membrane, and may also include corresponding cytoplasmic (intracellular) and extracellular loops. The seven transmembrane regions, as well as the extracellular and cytoplasmic loops, can be identified using standard methods, such as hydrophobic profiling, or methods described in Kyte & Doolittle, J. Mol. Biol., 157:105-32 (1982) or by Stryer. The general secondary and tertiary structures of transmembrane domains, particularly those of G protein-coupled receptors, such as the seven transmembrane domains of olfactory receptors, are known in the art. Therefore, the primary structural sequence can be predicted based on known transmembrane domain sequences, as detailed below. These transmembrane domains are useful for in vitro ligand-binding assays.

[0028] In the description of an assay for testing compounds that modulate OR family member-mediated olfactory transmission, the term "functional effect" includes the determination of any indirect or direct parameters under the influence of the receptor, such as functional, physical, and chemical effects. This includes ligand binding, changes in ion flux, membrane potential, current, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, signaling receptor-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP, IP3, or intracellular Ca3). 2+ ), including in vitro, in vivo, and ex vivo effects, and also including other physiological effects, such as increased or decreased neurotransmitter or hormone release.

[0029] In the description of an assay, "determination of functional effect" or "confirmation of activity" refers to an assay for compounds that increase or decrease indirect or direct parameters, such as functional, physical, and chemical effects, under the influence of OR family members. Such functional effects can be measured by any means known to those skilled in the art, such as changes in spectroscopic properties (e.g., fluorescence, absorbance, refractive index), hydrodynamics (e.g., shape), chromatography or solubility properties, patch clamp, voltage-sensitive dyes, total cell current, radioisotope efflux, induction markers, oocyte OR gene expression; tissue culture cell OR expression; transcriptional activation of OR genes or activity-inducing 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; and neurotransmitter release.

[0030] "Binders," "suppressors," "blockers," "inhibitors," and / or "modulators" of OR genes or proteins are interchangeable and refer to molecules identified using in vivo, in vitro, and ex vivo assays for olfactory transmission, such as ligands, agonists, antagonists, enhancers, and their homologs and mimetic forms, that bind, repress, block, inhibit, or modulate them.

[0031] Inhibitors are compounds that bind to a stimulus, partially or completely block the stimulus, reduce, suppress, prevent, delay, inactivate, desensitize, or downregulate olfactory transmission, such as antagonists. On the other hand, activators are compounds that bind to olfactory transmission, stimulate, increase, open up, promote, enhance, sensitize, or upregulate olfactory transmission, such as agonists.

[0032] Modulators include, for example, extracellular proteins that bind activators or inhibitors that also deactivate and desensitize the receptor (e.g., odor-binding proteins, ebnerin and other members of the hydrophobic carrier family, or members of the lipocalin family); G proteins; kinases (e.g., homologs of rhodopsin kinase and beta-adrenergic receptor kinase involved in receptor deactivation and desensitization); and compounds that alter the interaction with arrestin.

[0033] Modulators also include compounds that alter the affinity or transmission efficiency of ORs, thereby altering the effect of activators on ORs. Modulators may include, for example, genetically modified versions of OR family members with altered activity, as well as natural and synthetic ligands, antagonists, agonists, small chemical molecules, etc. Such assays for inhibitors and activators include, for example, the expression of OR family members in cells or cell membranes, the application of putative modulator compounds in or without the presence of flavor, fragrance, or odor molecules, such as odor-causing substances as defined herein, and the determination of the functional effect on olfactory transmission as described above. Samples or assays containing OR family members treated with potential activators, inhibitors, or modulators are compared to control samples without the inhibitor, activator, or modulator to test the degree of modulation. The control sample (not treated with a modifier) ​​is assigned a relative maximum OR activity value of 100%. OR inhibition is achieved when the OR activity level relative to the control is approximately 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 25-0%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0034] As used herein, the terms “purified,” “substantially purified,” and “isolated” refer to a state in which the compounds of the present invention are free from other different compounds that would normally associate with them in their natural state. Therefore, “purified,” “substantially purified,” and “isolated” substances include at least 0.5% by mass, 1% by mass, 5% by mass, 10% by mass, or 20% by mass, or at least 50% by mass or 75% by mass, of the mass of a given sample. In a particular embodiment, these terms refer to the compounds of the present invention including at least 95% by mass, 96% by mass, 97% by mass, 98% by mass, 99% by mass, or 100% by mass, of the mass of a given sample. As used herein, with respect to nucleic acids or proteins, the terms “purified,” “substantially purified,” and “isolated” nucleic acids or proteins also refer to a purified or concentrated state that differs from that which occurs naturally in the bodies of mammals, particularly humans. Any degree of purification or concentration greater than that which occurs naturally in the mammalian body, particularly in the human body, including (1) purification from other related structures or compounds, or (2) association with structures or compounds that do not normally associate in the mammalian body, particularly in the human body, is within the meaning of “isolated.” The nucleic acids or proteins or types of nucleic acids or proteins described herein may be isolated or associated with structures or compounds that do not otherwise normally associate in nature, according to various methods and processes known to those skilled in the art.

[0035] As used herein, the terms “amplifying” and “amplifying” refer to the use of any suitable amplification method to produce or detect recombination of naturally expressed nucleic acids, as described in detail below. For example, the present invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo-dT primers) for amplifying naturally expressed nucleic acids (e.g., genomic DNA or mRNA) or recombinant nucleic acids (e.g., cDNA) of the present invention (e.g., by polymerase chain reaction (PCR)).

[0036] The term "seven-transmembrane receptor" refers to polypeptides belonging to a superfamily of transmembrane proteins that have seven domains that transmembrane seven times across the plasma membrane (hence the seven domains are called "transmembrane" or "TM" domains, referred to as TM I to TM VII). Families of olfactory and certain gustatory receptors belong to this superfamily, respectively. Seven-transmembrane receptor polypeptides have similar characteristic primary, secondary, and tertiary structures, as will be discussed in more detail below.

[0037] The terms “nucleic acid” or “nucleic acid sequence” refer to deoxyribonucleotides or ribonucleotide oligonucleotides in single-stranded or double-stranded form. This term encompasses nucleic acids, i.e., oligonucleotides, including known analogues of natural nucleotides. It also encompasses nucleic acid-like structures with synthetic backings. Unless otherwise specified, a particular nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly shown sequences. Specifically, degenerate codon substitutions may be achieved, for example, by generating sequences in which the third position of one or more selected codons is substituted with a mixed base and / or a deoxyinosine residue.

[0038] It will be apparent to those skilled in the art that, in addition to the gene sequences shown in the sequences disclosed herein, variants may also include DNA sequence polymorphisms that may exist within a given population and may result in changes to the amino acid sequence of the polypeptide disclosed herein. Such genetic polymorphisms may exist in cells from different populations or in populations resulting from natural allelic mutations. Allelic variants may include functional equivalents.

[0039] Further embodiments also relate to molecules induced by such sequence polymorphisms from the nucleic acids specifically disclosed. These spontaneous variations typically result in variations of about 1–5% in the nucleotide sequence of the genes disclosed herein or the amino acid sequence of the polypeptides. As stated above, the nucleic acids encoding the polypeptides of the embodiments herein are useful means for modifying non-human host organisms or host cells intended for use in the methods described herein.

[0040] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues, whether or not they include naturally occurring and non-naturally occurring amino acids or polymers. When used in relation to a part of a nucleic acid, the term “heterogeneous” indicates that the nucleic acid contains two or more subsequences not found in nature in an identical relationship to one another. For example, a nucleic acid is typically produced by recombination and has two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another. Similarly, heterogeneous protein indicates that a protein contains two or more subsequences not found in nature in an identical relationship to one another (e.g., a fusion protein).

[0041] A “promoter” is defined as an array of nucleic acid sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the transcription start site, for example, the TATA element in the case of a polymerase type II promoter. Promoters may also optionally include distal enhancer or repressor elements, which may be located thousands of base pairs away from the transcription start site. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under the control of the environment or development. The term “operably linked” means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter or an array of transcription factor binding sites) and a second nucleic acid sequence, in which the expression regulatory sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0042] As used herein, “recombinant” means a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), a method for producing a gene product in a cell or other biological system using a recombinant polynucleotide, or a polypeptide encoded by a recombinant polynucleotide (“recombinant protein”). “Recombinant” also means the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources to an expression cassette or expression vector for the expression of a fusion protein containing the translocation domain of the present invention and a potentially amplified nucleic acid sequence using primers, e.g., inducible or constitutive expression. “Recombinant” also means modifications obtained by cell genome editing techniques, e.g., CRISPR / Cas9, resulting in stable or transient expression of endogenous genes, e.g., receptor genes as referred herein.

[0043] The term “expression vector” means any recombinant expression system intended for constitutive or inductive expression of the nucleic acid sequence of the present invention in vitro, ex vivo, or in vivo in any cell, including cells of prokaryotes, yeasts, fungi, plants, insects, or mammals. The term includes, without limitation, any linear or circular expression system, including viral vectors, bacteriophages, and plasmids. Those skilled in the art can select a suitable vector according to the expression system. The term includes expression systems that remain in the episome or are integrated into the host cell genome. Expression systems may have the ability to self-replicate or not self-replicate, i.e., have the ability to manipulate transient expression within a cell. The term includes recombinant “expression cassettes” that may contain the minimum elements necessary for transcription of recombinant nucleic acids. The term also includes, for example, genome editing methods, such as cassettes or vectors for the expression of endogenous genes via CRISPR / Cas9.

[0044] With respect to “non-human organism or host cell,” I mean a non-human organism or cell that includes and supports the replication or expression of the nucleic acid or expression vector described herein. The host cell may be a prokaryotic cell, e.g., Escherichia coli, or a eukaryotic cell, e.g., a yeast, insect, amphibian, or mammalian cell, e.g., CHO, HeLa, HEK-293, e.g., a cultured cell, an explant, or an in vivo cell.

[0045] A “tag” or “tag combination” refers to a short polypeptide sequence that can be attached to an odor receptor protein. Typically, the DNA encoding a “tag” or “tag combination” is attached to the DNA encoding the receptor, resulting in a fusion protein to which the “tag” or “tag combination” ultimately fuses at the N-terminus or C-terminus of the receptor. Lucy tags, FLAG® tags, and / or Rho tags can enhance receptor transport to the cell membrane and therefore support the expression of functional odor 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)].

[0046] "Geonol" and / or "Geosmin" refers to (4S,4aS,8aR)-4,8a-dimethyl-1,2,3,4,5,6,7,-octahydronaphthalene-4a-ol.

[0047] Specific Embodiments In particular, the present invention provides human olfactory receptors (ORs) OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2 as receptors for odor-causing substances such as geonol, dimethyl trisulfide (DMTS), 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, and transpyrrole (3-methyl-3-sulfanylhexanol), as shown in Table 1a. Use OR51I2, OR2H1, OR2W3, OR8G1, and their corresponding mouse orthologs Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, and Olfr558, as well as mouse ORs Olfr1487, Olfr339, Olfr1126, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46. [Table 1a]

[0048] Table 1b shows the corresponding sequence numbers of ORs applied according to the present invention: [Table 1b]

[0049] These receptors were not previously associated with odor-causing substances selected from geonol, dimethyl trisulfide (DMTS), 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, and transpyrrole.

[0050] In one embodiment, isolated nucleic acid molecules are provided herein that contain nucleic acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 or their reverse complements.

[0051] In one embodiment, the isolated nucleic acid sequences described herein are provided, which encode a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75.

[0052] In further embodiments, isolated polypeptides are provided herein that contain amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75.

[0053] In one embodiment, a non-human organism or host cell is transformed to express a polypeptide containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75.

[0054] In one embodiment, a non-human organism or host cell is transformed to express a polypeptide containing an amino acid sequence identical to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75.

[0055] Furthermore, this specification provides an expression vector comprising a nucleic acid encoding a polypeptide containing an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75.

[0056] This specification also provides an expression vector containing nucleic acids having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or their reverse complements.

[0057] In this specification, expression vectors are also provided that have nucleic acids containing nucleotide sequences identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or their reverse complements.

[0058] More specifically, the present invention relates to the following particular embodiments.

[0059] 1. A method for identifying compounds that bind, suppress, block, inhibit, and / or modulate the activity of olfactory receptors (ORs) activated by substances that cause laundry odors, mold odors, and / or sweat odors, a. OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, OR8G1, Olfr263, Olfr403, Olfr735, Olfr96, Olfr119 3, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322 and O Contacting at least one, particularly one olfactory receptor polypeptide selected from the group consisting of lfr46 and its chimeras or functional fragments; or at least one olfactory receptor polypeptide having at least 70%, for example 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with at least one, particularly one olfactory receptor polypeptide, with the test substance and substances that cause the odor of laundry, mold and / or sweat; and b. Measuring the response of at least one, particularly one, olfactory receptor to an odor-causing substance by measuring the response of the olfactory receptor in the presence and absence of the test substance. The method, including the method described above.

[0060] Certain subgroups of OR include the human ORs OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, and OR8G1.

[0061] Other specific subgroups of OR include the mouse ORs Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46.

[0062] 2. Furthermore, c. Identifying a test substance that modulates the response of at least one, particularly one, olfactory receptor, based on the response measured in the presence and absence of the test substance, and d. Select a test substance identified as a compound that modulates the response of at least one, particularly one, olfactory receptor to odor-causing substances. The method according to Embodiment 1, including the method described above.

[0063] 3. The method according to Embodiment 1 or 2, wherein the odor-causing substance is selected from the group consisting of geonol, dimethyl trisulfide (DMTS), 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, transpyrrole, and at least two, for example, mixtures of two or three of the above substances.

[0064] 4. The at least one olfactory receptor polypeptide, a. Containing an amino acid sequence having at least 70%, for example 75%, 80%, 85%, 90%, and especially 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75; and / or b. Encoded by nucleic acid molecules containing nucleotide sequences having at least 70%, for example 75%, 80%, 85%, 90%, and especially 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or their reverse complements, The method according to any of the above embodiments.

[0065] The specific subgroup of OR in this embodiment is sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 57, 59, or 61, or derived therefrom.

[0066] Other specific subgroups of OR in this embodiment are encoded by nucleic acid molecules containing the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 56, 58, 60 or nucleotide sequences derived therefrom.

[0067] 5. The polypeptide is a. Containing an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75; and / or b. Encoded by nucleic acid molecules containing nucleotide sequences having at least 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or their reverse complements, The method according to Embodiment 4.

[0068] The specific subgroup of OR in this embodiment is sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 57, 59, or 61, or derived therefrom.

[0069] Other specific subgroups of OR in this embodiment are encoded by nucleic acid molecules containing the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 56, 58, 60 or nucleotide sequences derived therefrom.

[0070] 6. The method according to any one of Embodiments 1 to 5, wherein the odor-causing substance is a laundry odor-causing substance selected from the group consisting of geonol, DMTS, 1-octen-3-ol, and a mixture of at least two of the said substances.

[0071] 7. The method according to any one of Embodiments 1 to 5, wherein the odor-causing substance is a mold odor-causing substance selected from the group consisting of geonol, DMTS, 1-octen-3-ol, and mixtures of at least two of the said substances.

[0072] 8. The method according to any one of Embodiments 1 to 5, wherein the odor-causing substance is a sweat odor-causing substance selected from the group consisting of butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, transpyrrole, and a mixture of at least two of the said substances.

[0073] 9. The olfactory receptor polypeptides are OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR4S2, Olfr263, Olfr403, Olfr735, Olfr1193, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr96, Olfr1322, Olfr93, Olfr398, Olfr1 The method according to Embodiment 6 or 7, selected from the group consisting of 20, Olfr1364 and Olfr937; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, for example 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides. The particular subgroup of OR in this embodiment is selected from OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, and OR4S2.

[0074] 10. The method according to Embodiment 9, wherein the odor-causing substance comprises 1-octen-3-ol, and the olfactory receptor is selected from the group consisting of OR2W1, OR1A1, OR2J3, OR5K1, OR4Q3, Olfr263, Olfr403, Olfr137, Olfr173, Olfr735, Olfr93, Olfr398, Olfr120, Olfr1364, and Olfr937; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, for example 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides. The specific subgroup of OR in this embodiment is selected from OR2W1, OR1A1, OR2J3, OR5K1, and OR4Q3.

[0075] 11. The method according to Embodiment 9, wherein the odor-causing substance comprises geonol, and the olfactory receptor is selected from the group consisting of OR1A1, OR11A1, OR2M3, Olfr403, Olfr164, Olfr1487, Olfr339, Olfr96, and Olfr1322; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, e.g., 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides. The specific subgroup of OR in this embodiment is selected from OR11A1, OR2M3, and OR1A1.

[0076] 12. The method according to Embodiment 9, wherein the odor-causing substance comprises DMTS, and the olfactory receptor is selected from the group consisting of OR2W1, OR1A1, OR5K1, OR4S2, Olfr263, Olfr1193, Olfr173, and Olfr403; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, e.g., 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides. The specific subgroup of OR in this embodiment is selected from OR2W1, OR1A1, OR5K1, and OR4S2.

[0077] 13. The method according to Embodiment 8, wherein the olfactory receptor polypeptide is selected from the group consisting of OR2W1, OR1A1, OR2J3, OR5K1, OR51E1, OR51I2, Olfr263, Olfr403, Olfr641, Olfr137, Olfr173, Olfr1126, Olfr558 and Olfr46; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, for example 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides. The specific subgroup of OR in this embodiment is selected from OR2W1, OR1A1, OR2J3, OR5K1, OR51E1 and OR51I2.

[0078] 14. The method according to Embodiment 13, wherein the odor-causing substance contains butyric acid, and the olfactory receptor is selected from the group consisting of OR51E1 and Olfr558; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, e.g., 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides. The particular subgroup of OR in this embodiment is OR51E1.

[0079] 15. The method according to Embodiment 13, wherein the odor-causing substance comprises 3-methyl-2-hexenoic acid, and the olfactory receptor is selected from the group consisting of OR51I2, OR2W1, Olfr641 and Olfr263; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, for example 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides.

[0080] 16. The method according to Embodiment 13, wherein the odor-causing substance comprises 3-hydroxy-3-methylhexanoic acid, and the olfactory receptor is selected from the group consisting of OR2W1, Olfr1126 and Olfr263; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, for example 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides.

[0081] 17. The method according to Embodiment 13, wherein the odor-causing substance comprises transpyrrole, and the olfactory receptor is selected from the group consisting of OR2W1, OR1A1, OR2J3, OR5K1, Olfr263, Olfr137, Olfr173, Olfr403, and Olfr46; their chimeras and functional fragments; and olfactory receptor polypeptides having at least 70%, e.g., 75%, 80%, 85%, 90%, and particularly 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to at least one of the olfactory receptor polypeptides. The specific subgroup of OR in this embodiment is selected from OR2W1, OR1A1, OR2J3, and OR5K1.

[0082] 18. A method for identifying compounds that bind, suppress, block, inhibit, and / or modulate the activity of at least one olfactory receptor activated by an odor-causing substance, The aforementioned receptor, a. Containing an amino acid sequence having at least 70%, for example 75%, 80%, 85%, 90%, and especially 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75; and / or b. Encoded by nucleic acid molecules containing nucleotide sequences having at least 70%, for example 75%, 80%, 85%, 90%, and especially 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or their reverse complements, It is a polypeptide, The method described above is 1. Contacting a receptor, or a chimera or fragment thereof, with a compound; 2. To determine whether the compound has an effect on the receptor's activity. including and The method wherein the odor-causing substance is selected from the group consisting of geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, transpyrrole, and a mixture of at least two of the said substances.

[0083] The specific subgroup of OR in this embodiment is sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 57, 59, or 61, or derived therefrom.

[0084] Other specific subgroups of OR in this embodiment are encoded by nucleic acid molecules containing the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 56, 58, 60 or nucleotide sequences derived therefrom.

[0085] 19. A method for identifying compounds that bind, suppress, block, inhibit, and / or modulate the activity of olfactory receptors activated by substances that cause laundry odor, mold odor, and / or sweat odor, selected from the group consisting of geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, transpyrrole, and mixtures of at least two of the said substances, (i) OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, OR8G1, Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, O Contacting a cell line expressing at least one olfactory receptor polypeptide selected from the group consisting of lfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46 and their chimeric or functional fragments, or at least one olfactory receptor polypeptide having at least 70% amino acid sequence identity with at least one of the said olfactory receptor polypeptides, with at least one compound. (ii) Screening for compounds that bind, inhibit, block, block, and / or modulate the activity of the olfactory receptor polypeptide, and (iii) Identify compounds that presumptively modulate laundry odor, mold odor and / or sweat odor by binding to, suppressing, blocking, inhibiting, and / or modulating the activity of the receptor polypeptide. The method, including the method described above.

[0086] Certain subgroups of OR include OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, and OR8G1.

[0087] Other specific subgroups of OR are Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46.

[0088] 20. Isolated polypeptides containing amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75.

[0089] 21. An isolated nucleic acid molecule containing a nucleic acid sequence encoding the polypeptide described in Embodiment 20.

[0090] 22. An isolated nucleic acid molecule according to Embodiment 20, comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or their reverse complements.

[0091] 23. Recombinant nucleic acid molecules, a. Nucleic acids comprising a combination of tags including at least one of the Lucy tag, FLAG® tag, and / or Rho tag; and b. Nucleic acids encoding receptors selected from the group consisting of OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, OR8G1, Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46, or their complements. Recombinant nucleic acid molecules, including those containing this molecule.

[0092] The specific subgroups of OR are OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, and OR8G1.

[0093] Other specific subgroups of OR are Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46.

[0094] 24. The recombinant nucleic acid molecule according to Embodiment 23, wherein the Lucy tag contains SEQ ID NO: 51, the FLAG® tag contains SEQ ID NO: 47, and the Rho tag contains SEQ ID NO: 49.

[0095] 25. An expression vector comprising the nucleic acid described in any of Embodiments 21 to 24.

[0096] 26. A non-human host organism or host cell that has been modified to express a receptor selected from the group consisting of OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, OR8G1, Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46.

[0097] The specific subgroups of OR are OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, and OR8G1.

[0098] Other specific subgroups of OR are Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46.

[0099] 27. The non-human host organism or host cell according to Embodiment 26, wherein the receptor comprises a polypeptide described in Embodiment 19, or a polypeptide encoded by a nucleic acid described in any of Embodiments 21 to 24.

[0100] 28. A non-human host organism or host cell, a. A nucleic acid according to any one of embodiments 21 to 24, or b. Expression vector according to Embodiment 25 Non-human host organisms or host cells, including those mentioned above.

[0101] 29. A non-human host organism or host cell according to any one of embodiments 26 to 28, wherein the cell is a eukaryotic cell.

[0102] 30. A non-human host organism or host cell according to any one of embodiments 26 to 28, wherein the cell is a prokaryotic cell.

[0103] 31. A non-human host organism or host cell according to any one of embodiments 26 to 29, selected from the group consisting of cells derived from HEK293, CHO, African clawed frog (Xenopus oocytes), COS, yeast, and olfactory placode.

[0104] 32. Use of polypeptides that can be activated by substances selected from the group consisting of geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, transpyrrole, and mixtures of at least two of the said substances, for the identification of malodorous compounds.

[0105] The specific subgroup of OR in this embodiment is sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or derived therefrom.

[0106] As shown herein, the ORs identified according to the present invention may be specific to a single MCS or activated by a panel of structurally different MCSs. Therefore, the present invention enables different strategies for identifying suitable modifiers.

[0107] For example, if the target is an inhibitor of Geonol only, it may be prudent to use OR11A1 or OR2M3 in the assay method of the present invention.

[0108] For example, when inhibitors of DTMS, 1-octen-3-ol, and optionally geonol are being investigated, it may be prudent to use OR2W3, OR5K1, or optionally OR1A1 in the assay method of the present invention. Other assay strategies for identifying modifiers that neutralize substances causing laundry odor, mold odor, or sweat odor, or for identifying modifiers that neutralize substances causing laundry odor, mold odor, and sweat odor, will become apparent from the experimental results disclosed herein.

[0109] Furthermore, there are provided one of a number of odor-modulating compounds identified by methods disclosed herein, which bind, suppress, block, inhibit, and / or modulate the activity of olfactory receptors activated by odor-causing substances selected from the group consisting of geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, transpyrrole, and mixtures of at least two of these substances.

[0110] According to one embodiment, OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, OR8G1, Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487 The following are provided: odor-modulating compounds that bind, inhibit, block, block, and / or modulate the activity of at least one olfactory receptor selected from the group consisting of Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46, and are identified by the methods disclosed herein. The particular subgroup of ORs in this embodiment is OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, and OR8G1, or derived therefrom. Other specific subgroups of OR are Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, Olfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46.

[0111] The test substance to be tested by the method of the present invention is not particularly limited. The test substance may be a naturally occurring substance or a chemically or biologically synthesized artificial substance. The test substance may be a compound or a mixture of compounds.

[0112] To identify unknown receptors specific to MCS compounds such as geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole, isolated olfactory neurons (OSNs) can be screened using the aforementioned MCS compounds. The MCS compounds can further be used in cell-based dose-response experiments performed with the MCS-specific receptors to evaluate both receptor specificity and sensitivity.

[0113] In one embodiment, the Specified provides a method for identifying mammalian odor receptors for malodorous compounds, and the use of receptors for screening, particularly for high-throughput screening (HTS) of malodorous compounds (e.g., those that bind, suppress, block, inhibit and / or modulate the activity of ORs).

[0114] Method for monitoring OR activity OR may be used in any form in the method of the present invention, provided that the function of OR is not impaired. For example, OR may be used as follows: OR isolated from living organisms and their culture products, e.g., tissues or cells that essentially express olfactory neurons; olfactory cell membranes having OR; recombinant cells genetically modified to express OR and their culture products; recombinant cell membranes; and artificial lipid bilayers having OR.

[0115] In further embodiments, indicators for monitoring olfactory receptor activity are selected from fluorescent calcium indicator dyes, calcium indicator proteins (e.g., GcaMP, a genetically encoded calcium indicator), fluorescent cAMP indicators, cell recruitment assays, cell dynamic mass redistribution assays, label-free cell-based assays, cAMP response element (CRE)-mediated reporter proteins, biochemical cAMP HTRF assays, beta-arrestin assays, or electrophysiological recordings. In particular, calcium indicator dyes (e.g., Fura-2 AM) that can be used to monitor the activity of olfactory receptors expressed on the membranes of olfactory neurons are selected.

[0116] In a specific embodiment, compounds are screened sequentially, and the odor-dependent changes in calcium dye fluorescence are measured using a fluorescence microscope or a fluorescence-excited cell sorter (FACS).

[0117] In further embodiments, molecular 3D receptor modeling of olfactory receptors is used to evaluate in silico binding ability and to identify compounds that can activate, mimic, block, inhibit, modulate, and / or enhance the activity of olfactory receptors.

[0118] For example, olfactory neurons activated with geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole are isolated using glass capillaries attached to a micromanipulator or FACS instrument. Mouse olfactory neurons are isolated using Ca in the same manner as described above. 2+Screening is performed by imaging [Malnic, B. et al. Cell 96, 713-723 (1999); Araneda, RC et al. J. Physiol. 555, 743-756 (2004); and International Publication No. 2014 / 210585, incorporated herein by reference]. In particular, the use of an electric mobile microscope stage increases the number of cells that can be screened to at least 1500 per experiment. Since there are approximately 1200 different olfactory receptors in mice, and each olfactory neuron expresses only one of the 1200 olfactory receptor genes, this screening capability covers substantially the entire mouse odor receptor repertoire. In other words, the combination of calcium imaging for high-throughput olfactory neuron screening leads to the identification of almost all odor receptors that respond to specific profiles of odorants. In certain embodiments, odor receptors responding to geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole can be isolated. For example, at least one neuron can be isolated.

[0119] For calcium imaging of olfactory neurons, the main olfactory epithelium may be dissected from the mouse before neuronal dissociation. The dissected olfactory epithelium may then be transferred to a dissociation buffer for mechanical and enzymatic dissociation. The dissociated neurons may then be seeded on coverslips to allow screening of thousands of cells by fluorescence microscopy, and the cells may be loaded with a calcium-sensitive dye (Fura-2 AM) for approximately 30 minutes at, for example, 31°C, and then transferred to a microscope ready for screening. The cells are stimulated by perfusing a diluted solution of odorants (in physiological saline) over the dissociated olfactory neurons. Sparse cells responsive to malodorous compounds are stimulated by stimulating their receptors with, for example, 50 μM of the malodorous compound, and intracellular Ca is indicated by a change in Fura-2 fluorescence. 2+Identification is performed by monitoring the flow. After analysis, responsive cells may be collected from glass coverslips using a suction micropipette. The isolated cells are then pooled into a single sample or processed individually for identification of odor receptor genes expressed as mRNA in the responsive cells.

[0120] In certain embodiments, olfactory neuron mRNA is purified and amplified according to a method commonly described by Marko, NF et al. ((2005) A robust method for the amplification of RNA in the sense orientation. BMC genomics, 6, 27; doi:10.1186 / 1471-2164-6-27 (Eberwine method)). At least a portion of the transcriptome (including the entire transcriptome) is sequenced using next-generation sequencing (NGS) technology or hybridized to known genes using microarray technology. NGS is commonly discussed and described in Metzker, ML Nat. Rev. Genet. 11, 31-46 (2010). In certain embodiments, at least five neurons presenting identical response profiles are pooled. mRNA is released by cell lysis immediately after picking; no DNAse and purification steps are performed. mRNA is amplified by two consecutive in vitro transcriptions (IVT). Amplification may be performed according to the MessageAmpII aRNA kit (Ambion, AMA1751) using the following parameters: two rounds of continuous 14-hour IVT.

[0121] In a further embodiment, mRNA from a single olfactory neuron is purified and amplified by an LD-PCR (long-range polymerase chain reaction) based method, such as those described in an NGS-ready kit (e.g., Clontech / Takara, SMARTer® Ultra® Low Input RNA Kit-v3 for sequencing, cat.634848). The single-cell mRNA is first reverse transcribed to the corresponding cDNA, then amplified for 18 PCR cycles, and provided as an NGS sample for transcriptome sequencing.

[0122] In further embodiments, the identity of olfactory receptor groups or gene families for geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole is determined by comparing the results of NGS reads obtained from isolated activated olfactory neurons with the same type of reference genome sequence (e.g., up to the number of neurons collected). In particular, the putative receptors for geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole are the most abundant olfactory receptor mRNAs present in NGS samples derived from olfactory neurons or in more than one independent biological replication. Due to the nature of the olfactory code combinations (one compound activates many ORs, and one OR is activated by many compounds), a pool of several neurons activated by a given compound allows for the search of substantially all receptors involved in the perception of these molecules in a single NGS experiment. Therefore, a pool of functionally similar neurons significantly improves the throughput and speed of de-orphanization.

[0123] Next, using standard bioinformatics tools, we identify the human odor receptor most closely related to other putative mammalian (non-human) receptors for geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole, under the assumption that homologous sequence receptors retain similar function. Several methods correctly identify human OR ligand pairs based on this assumption [Armelin-Correa and Malnic (2017)], and up to 80% of mouse-human orthologs appear to maintain similar functional response profiles [Adipietro, K. A, et al., PLoS Genet. 8, e1002821-e1002821 (2012)]. Default parameters of the BLASTP and / or BLASTN algorithms, or other ortholog pair identification algorithms, such as InParanoid, may be used.

[0124] Human or non-human mammalian receptors for geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole may be adapted to functional assays that can be used to identify compounds that bind, repress, block, inhibit, and / or modulate the activation of olfactory receptors. In particular, the assay may be a cell-based assay or a binding assay, and the method for identifying the compounds may be a high-throughput screening assay. More specifically, receptor-based assays are provided herein that are adaptable to high-throughput screening of receptors in a compound library for the discovery of compounds modulating (e.g., binding, block, inhibiting, repressing, and masking) compounds.

[0125] In certain embodiments, receptor gene sequences for geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole are identified from cells sensitive to geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole as follows: Pooled neurons are heated at 75°C for 10 minutes to disrupt the cell membrane and make their mRNA available for amplification. This amplification step is important when applying NGS technology with a limited amount of starting material, typically 1 to 15 cells. Linear amplification according to the Eberwine method (IVT) ensures the maintenance of the relative transcription levels of the expressed gene. Two consecutive in vitro transcriptions are used overnight (14 hours) to obtain a sufficient amount of cRNA; and the amplified cRNA is then used to create an Illumina HiSeq cDNA library. The resulting short sequences, typically 75–150 base pairs long (commonly called "reads"), are aligned against a mouse reference genome (e.g., UCSC version mm9 or mm10) to construct a complete transcriptome of these cells. Quantitative analysis of the transcriptome data yields a list of transcribed odor receptor genes and their respective expression levels. Odor receptor genes showing the most abundant levels of mRNA (most abundant "reads") or present in more than one repeated experiment are considered to be receptors for putative geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole.

[0126] Next, using the predicted mouse OR gene, the latest versions of both mouse and human genome databases are mined to identify the most closely related receptors (i.e., those with the highest sequence similarity) in mouse (paralogus gene) and human (orthologus gene). This process may be performed using the BLAST search algorithm (published on the NCBI website), a sequence similarity search tool, which uses all previously obtained putative gene sequences from the initial transcriptome analysis as query sequences. The newly identified genes identified from this data mining process are considered to be potential receptors for geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole, under the assumption that the paralogus and orthologus genes are likely to exhibit similar activity. In certain embodiments, pairwise comparisons of sequence homology are performed to identify closely related receptors in mouse and human, and then identify the receptors as described in International Publication No. 2014 / 210585. Other approaches, such as RT-PCR and microarray approaches, may also be used.

[0127] In a further embodiment, to complete the de-orphanization process, candidate OR genes are further expressed in vitro to confirm their activity against the compounds used, and olfactory neurons and other structurally relevant compounds of interest are isolated. Mouse receptors identified from isolated olfactory neurons responding to geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole are modified at their N-terminus with short polypeptide sequences (e.g., FLAG® tag (SEQ ID NO: 48), Rho tag (SEQ ID NO: 50; the first 20 amino acids of the bovine rhodopsin receptor), and / or Lucy tag (SEQ ID NO: 52; a cleavable leucine-rich signal peptide sequence), and HEK Transient expression is performed in 293T cells, and then individual stimulation with geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole is performed to confirm their identity as receptors for genuine geonol, dimethyl trisulfide (DMTS), 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole. In a further embodiment, the RTP1 gene can also be expressed in cell lines, whether by activation of the endogenous RTP1 gene or by transformation. Human G alpha subunit Gα in this cell-based assay olf Co-expression of the human G alpha subunit Gα activates the Gs signaling pathway, which, upon binding to the appropriate ligand, leads to an increase in internal cAMP. Alternatively, in cell-based assays, human G alpha subunit Gα 15 The simultaneous expression of the internal Ca when it binds to the appropriate ligand 2+ This activates the Gq signaling pathway, which leads to an increase in [a certain factor]. The process and the results obtained so far are useful in verifying a process for the rapid and reliable identification of mammalian odor receptors for geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole.

[0128] Furthermore, assays are provided for identifying compounds that bind to odor receptors of geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole. In further embodiments, assays are provided for identifying compounds that bind to odor receptors of OR2W1, OR1A1, OR2J3, OR4Q3, OR5K1, OR11A1, OR2M3, OR51E1, OR4S2, OR51I2, OR2H1, OR2W3, OR8G1, Olfr263, Olfr403, Olfr735, Olfr96, Olfr1193, Olfr641, Olfr137, Olfr173, Olfr164, Olfr1487, Olfr339, O Malodor-modulating compounds, such as those listed below, are provided herein, which bind, inhibit, block, block, and / or modulate the activity of at least one olfactory receptor selected from the group consisting of lfr1126, Olfr558, Olfr93, Olfr398, Olfr120, Olfr1364, Olfr937, Olfr1322, and Olfr46, and are identified by the methods described herein.

[0129] In one embodiment, the activation of the compound is measured by comparing its binding to the activation of geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole. In another embodiment, a receptor or a chimera or fragment thereof is brought into contact with the compound in the presence of geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole, under conditions that allow the compound to bind to the receptor together with geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole.

[0130] In a further embodiment, the compound is brought into contact with receptors activated by geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole, or chimeric or fragments thereof, and the receptors, or chimeric or fragments thereof, are expressed in cells that have been recombinantly modified to express receptor polypeptides.

[0131] The activity of compounds can be measured in vivo, ex vivo, in vitro, and using synthetic screening systems.

[0132] In other embodiments, contact is carried out using liposomes or virus-induced budding membranes containing the polypeptides described herein.

[0133] In other embodiments, methods for identifying compounds that bind, inhibit, block, block, and / or modulate the activity of olfactory receptors that can be activated by geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole may be carried out in intact cells or in membrane fractions from cells expressing the polypeptides described herein.

[0134] Therefore, the ORs described herein constitute candidate receptors of value for the identification of modulators, antagonists, and / or blockers that are involved in the perception of malodors induced by geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole, and modulate, reduce, suppress, inhibit, and / or block the perception of laundry malodors, mold malodors, and / or sweat malodors.

[0135] Polypeptides applicable according to the present invention The present invention relates to "functional equivalents," "analogs," or "functional variants" of polypeptides specifically described herein.

[0136] For example, a "functional equivalent" refers to a polypeptide that exhibits an OR activity that is at least 1-10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% higher or lower than that defined herein in the test used for OR activity.

[0137] The "functional equivalents" according to the present invention also include specific mutants having amino acids that are different from the specifically mentioned amino acids but exhibit one of the aforementioned biological activities at at least one sequence position of the amino acid sequences listed herein. Thus, "functional equivalents" include mutants obtained by addition, substitution, particularly conservative substitution, deletion, and / or inversion of one or more, for example, 1-20, 1-15, or 5-10 amino acids, and the changes mentioned can occur at any sequence position as long as they lead to mutants having the profile of properties according to the present invention. Functional equivalents are provided particularly when the activity pattern is qualitatively identical between the mutant and the unaltered polypeptide, i.e., when the interaction with, for example, the same agonist or antagonist occurs at different rates (i.e., when represented by EC 50 or IC 50 values or when represented by other parameters suitable for the art). Examples of suitable (conservative) amino acid substitutions are shown in the following table:

Chemical formula

[0138] The "functional equivalents" in the above sense are also the "precursors" of the described polypeptides, as well as the "functional derivatives" and "salts" of the polypeptides.

[0139] A "precursor" is, in that case, a natural or synthetic precursor of a polypeptide that may or may not have the desired biological activity.

[0140] The term "salt" in this invention refers to salts of the carboxyl group and salts of the amino group obtained by acid addition of the protein molecule according to this invention. Salts of the carboxyl group may be produced by known methods and include inorganic salts, such as sodium, calcium, ammonium, iron, and zinc salts, as well as salts with organic bases, such as amines, such as triethanolamine, arginine, lysine, and piperidine. Salts of acid addition, such as salts with inorganic acids, such as hydrochloric acid or sulfuric acid, and salts with organic acids, such as acetic acid and oxalic acid, are also included in this invention.

[0141] The "functional derivatives" of polypeptides according to the present invention may be produced using known techniques on or at the N-terminus or C-terminus of functional amino acids. Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtained by reaction with ammonia or primary or secondary amines; N-acyl derivatives of free amino groups produced by reaction with acyl groups; or O-acyl derivatives of free hydroxyl groups produced by reaction with acyl groups.

[0142] "Functional equivalents" naturally include polypeptides obtained from other organisms, as well as naturally occurring variants. For example, the range of homologous sequence regions may be established by sequence comparison, and the equivalent polypeptide may be determined based on the specific parameters of the present invention.

[0143] "Functional equivalents" include, for example, fragments of the polypeptide according to the present invention that exhibit a desired biological function, preferably individual domains or sequence motifs.

[0144] A “functional equivalent” is a fusion protein having one of the polypeptide sequences or functional equivalents derived therefrom as described herein and at least one further functionally distinct heterologous sequence in a functional N-terminal or C-terminal association (i.e., without substantial mutual dysfunction of the fusion protein portion). Examples of these heterologous sequences, not limited to these, include signal peptides, histidine anchors, or enzymes.

[0145] Similarly, “functional equivalents” included in accordance with the present invention are homologs of the specifically disclosed polypeptides. These have at least 60%, preferably at least 75%, particularly at least 80% or 85%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology (or identity) to one of the specifically disclosed amino acid sequences, calculated by the Pearson and Lipman algorithm (Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448). The homology or identity expressed as a percentage of homologous polypeptides according to the present invention means identity expressed as a percentage of amino acid residues based on the full length of one of the amino acid sequences specifically described herein.

[0146] Identity data, expressed as a percentage, may be determined using BLAST alignment, the blastp (protein-protein BLAST) algorithm, or by applying the Clustal settings specified below herein.

[0147] In the case of possible protein glycosylation, the “functional equivalents” according to the present invention include polypeptides described herein in the form of deglycosylation or glycosylation, and in modified forms obtained by altering the glycosylation pattern.

[0148] Such functional equivalents or homologs of polypeptides according to the present invention may be generated by mutagenesis, for example, by point mutation, elongation or shortening of proteins, or as described in more detail below.

[0149] Such functional equivalents or homologs of polypeptides according to the present invention can be identified by screening combinatorial databases of mutants, for example, truncated mutants. For example, diverse databases of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, for example, by enzymatic ligation of a mixture of synthetic oligonucleotides. There are many methods that can be used to generate a database of potential homologs from degenerate oligonucleotide sequences. Degenerate gene sequences may be chemically synthesized using an automated DNA synthesizer, and the synthetic genes may be ligated into a suitable expression vector. The use of a degenerate genome makes it possible to provide all sequences in a mixture and encode a desired set of potential protein sequences. Methods for synthesizing degenerate oligonucleotides are known to those skilled in the art (e.g., Narang, SA (1983); Itakura et al. (1984)(a); Itakura et al. (1984)(b); Ike et al. (1983)).

[0150] In the prior art, several techniques are known for screening gene products of combinatorial databases generated by point mutations or shortenings, and for screening cDNA libraries of gene products having selected characteristics. These techniques can be adapted for rapid screening of gene banks generated by combinatorial mutagenesis of homologs according to the present invention. The most frequently used techniques for screening large gene banks based on high-throughput analysis include cloning the gene bank with replicable expression vectors, transforming suitable cells in the resulting vector database, and expressing the combined gene under conditions where detection of desired activity facilitates the isolation of the vector encoding the detected gene. Recurrent ensemble mutation (REM), a technique that increases the frequency of functional variants in the database, can be used in combination with screening tests to identify homologs (Arkin and Yourvan (1992); Delgrave et al. (1993)).

[0151] Applicable coding nucleic acid sequences according to the present invention The present invention also relates to nucleic acid sequences encoding polypeptides as defined herein.

[0152] The present invention also relates to nucleic acids having a certain degree of "identity" with the sequences specifically disclosed herein. The "identity" between the two nucleic acids means the identity of the nucleotides throughout the entire length of the nucleic acid in each case.

[0153] For example, identity can be calculated using the Vector NTI Suite 7.1 program from Informax (USA), which employs the Clustal Method (Higgins DG, Sharp PM. (1989)), with the following settings: [ka]

[0154] Alternatively, identity may be measured according to the webpage of Chenna et al. (2003): http: / / www.ebi.ac.uk / Tools / clustalw / index.html# and with the following settings: [ka]

[0155] All nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, e.g., cDNA and mRNA) listed herein can be produced by chemical synthesis from nucleotide components, for example, by degeneracy of individual overlapping complementary nucleic acid components of a double helix, as is known. Chemical synthesis of oligonucleotides can be carried out by known methods, for example, the amidite phosphite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and gap filling by Klenow fragment and ligation reactions of DNA polymerase, as well as general cloning techniques, are described in Sambrook et al. (1989) (see below).

[0156] The present 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 polypeptides and their functional equivalents obtained, for example, using artificial nucleotide analogs.

[0157] The present invention relates to both isolated nucleic acid molecules encoding polypeptides or biologically active segments thereof according to the present invention, and nucleic acid fragments that can be used as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the present invention, for example.

[0158] The nucleic acid molecule according to the present invention may further include untranslated sequences from the 3' and / or 5' ends of the coding gene region.

[0159] The present invention further relates to nucleic acid molecules that are complementary to a nucleotide sequence or segment thereof as specifically described.

[0160] The nucleotide sequences according to the present invention enable the generation of probes and primers that can be used for the identification and / or cloning of homologous sequences in other cell types and organisms. Such probes or primers generally include a nucleotide sequence region that hybridizes under "stringent" conditions (see below) with at least about 12, preferably at least about 25, e.g., about 40, 50, or 75 consecutive nucleotides of the sense strand or corresponding antisense strand of the nucleic acid sequence according to the present invention.

[0161] "Isolated" nucleic acid molecules are separated from other nucleic acid molecules present in the natural source of nucleic acids, and furthermore, if produced by recombinant technology, they do not require substantially other cell material or culture medium, or if chemically synthesized, they do not require chemical precursors or other chemical substances.

[0162] Nucleic acid molecules according to the present invention can be isolated by standard molecular biology techniques and sequence information provided in accordance with the present invention. For example, cDNA can be isolated from a suitable cDNA library using one of the specifically disclosed whole sequences or a segment thereof as a hybridization probe and standard hybridization techniques (e.g., described in Sambrook (1989)). Furthermore, nucleic acid molecules containing one of the disclosed sequences or a segment thereof can be isolated by polymerase chain reaction using oligonucleotide primers constructed based on this sequence. The thus amplified nucleic acid is cloned into a suitable vector and characterized by DNA sequencing. Oligonucleotides according to the present invention can also be produced by standard synthetic methods, for example, using an automated DNA synthesizer.

[0163] Nucleic acid sequences or derivatives thereof, homologs or parts thereof, according to the present invention can be isolated, for example, by conventional hybridization techniques or PCR techniques from other bacteria, for example, by decomposing a genome or cDNA library. These DNA sequences hybridize with the sequences according to the present invention under standard conditions.

[0164] "Hybridizing" refers to the ability of polynucleotides or oligonucleotides to bind to nearly complementary sequences under standard conditions; however, non-specific binding does not occur between non-complementary partners under these conditions. Therefore, sequences may be 90-100% complementary. The property of complementary sequences to bind specifically to each other is utilized, for example, in Northern blotting or Southern blotting, or in primer binding in PCR or RT-PCR.

[0165] Short oligonucleotides of conserved regions are advantageously used for hybridization. However, it is also possible to use longer fragments of nucleic acids or the entire sequence for hybridization according to the present invention. These standard conditions vary depending on the nucleic acid used (oligonucleotide, longer fragment, or entire sequence) or the type of nucleic acid used for hybridization (DNA or RNA). For example, the melting temperature for DNA:DNA hybrids is about 10°C lower than that of DNA:RNA hybrids of the same length.

[0166] For example, depending on the specific nucleic acid, standard conditions mean 42-58°C in a buffered aqueous solution at a concentration of 0.1-5×SSC (1×SSC = 0.15M NaCl, 15mM sodium citrate, pH 7.2) or at a temperature of 42-58°C in the presence of 50% formamide, e.g., 42°C in 5×SSC and 50% formamide. Advantageously, hybridization conditions for DNA:DNA hybrids are 0.1×SSC and a temperature of approximately 20°C-45°C, preferably approximately 30°C-45°C. Advantageously, hybridization conditions for DNA:RNA hybrids are 0.1×SSC and a temperature of approximately 30°C-55°C, preferably approximately 45°C-55°C. These aforementioned hybridization temperatures are examples of melting temperature values ​​calculated for nucleic acids having a length of approximately 100 nucleotides and a G+C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant genetics textbooks, e.g., Sambrook et al. (1989), and can be calculated using formulas known to those skilled in the art, depending on factors such as nucleic acid length, hybrid type, or G+C content. Those skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds) (1985), Brown (ed) (1991).

[0167] Hybridization can be performed under particularly stringent conditions. Such hybridization conditions are described, for example, in Sambrook (1989) or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6.

[0168] As used herein, the terms hybridization or hybridize under specific conditions are intended to refer to hybridization and washing conditions in which nucleotide sequences that are significantly identical or homologous to each other remain bound to one another. Such conditions may be those in which sequences that are at least about 70%, for example, at least about 80%, and at least about 85%, 90%, or 95% identical remain bound to one another. Definitions of low stringency, moderate, and high stringency hybridization conditions are provided herein.

[0169] Appropriate hybridization conditions can be selected by those skilled in the art with minimal experiments, as described in Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Furthermore, stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0170] As used herein, the defined conditions for low stringency are as follows: A filter containing DNA is pretreated at 40°C for 6 hours in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is carried out in the same solution with the following modifications: using 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (wt / vol) dextransphosphate, and a 5-20 × 10⁶ 32P labeled probe. The filter is incubated in the hybridization mixture at 40°C for 18-20 hours and then washed at 55°C for 1.5 hours. Wash in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. Replace the washing solution with a fresh solution and incubate at 60°C for a further 1.5 hours. Absorb and dry the filter and expose it for autoradiography.

[0171] As used herein, the defined conditions for moderate stringency are as follows: A filter containing DNA is pretreated at 50°C for 7 hours in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is carried out in the same solution with the following modifications: using 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (wt / vol) dextransphosphate, and a 5-20 × 10⁶ 32P labeled probe. The filter is incubated in the hybridization mixture at 50°C for 30 hours and then washed at 55°C for 1.5 hours. Wash in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. Replace the washing solution with a fresh solution and incubate at 60°C for a further 1.5 hours. Absorb and dry the filter and expose it for autoradiography.

[0172] As used herein, the defined conditions for high stringency are as follows: Pre-hybridization of the DNA-containing filter is performed at 65°C for 8 hours to overnight in a buffer consisting of 6×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. The filter is hybridized at 65°C for 48 hours in a pre-hybridization mixture containing 100 μg / ml denatured salmon sperm DNA and 5 to 20 × 10⁶ cpm of 32P-labeled probe. The filter is washed at 37°C for 1 hour in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. Subsequently, it is washed at 50°C for 45 minutes with 0.1×SSC.

[0173] Other less common, moderate, and high stringency conditions known in the art (e.g., those used in interspecific hybridization) may be used when the aforementioned conditions are unsuitable (e.g., when used in interspecific hybridization).

[0174] The present invention also relates to a nucleic acid sequence specifically disclosed or a derivative of an inducible nucleic acid sequence.

[0175] Therefore, further nucleic acid sequences according to the present invention may be derived from the sequences specifically disclosed herein and may differ therefrom by the addition, substitution, insertion or deletion of individual nucleotides or several nucleotides, and may further encode polypeptides having a profile of desired properties.

[0176] The present invention also includes nucleic acid sequences that contain or are modified with so-called silent mutations compared to the specifically cited sequences, according to the codon usage of specific protozoa or host organisms and their naturally occurring variants, such as splicing variants or allele variants.

[0177] This also concerns sequences obtained by conservative nucleotide substitutions (i.e., the amino acid in question is replaced by an amino acid of the same charge, size, polarity, and / or solubility).

[0178] The present invention also relates to molecules derived from nucleic acids specifically disclosed by sequence polymorphisms. These gene polymorphisms may exist among individuals within a population due to natural variations. These natural variations typically result in 1-5% variation in the nucleotide sequence of a gene.

[0179] The nucleic acid sequence derivatives according to the present invention mean allelic variants having, for example, at least 60% homology at the derived amino acid level, preferably at least 80% homology across the entire sequence range, and very preferably at least 90% homology (for homology at the amino acid level, see the details provided above for polypeptides). Advantageously, the homology may be higher than that of a subregion of the sequence.

[0180] Furthermore, derivatives should also be understood as homologs of nucleic acid sequences according to the present invention, such as homologs of animals, plants, fungi, or bacteria, truncated sequences, coding and non-coding DNA sequences, and single-stranded DNA or single-stranded RNA. For example, homologs have at least 40%, preferably at least 60%, particularly preferably at least 70%, and very particularly preferably at least 80% homology at the DNA level across a given DNA region in the sequence specifically disclosed herein.

[0181] Furthermore, derivatives should be understood, for example, as fusion products with promoters. Promoters attached to the given nucleotide sequences may be modified by at least one nucleotide exchange, at least one insertion, inversion, and / or deletion, without impairing the functionality or efficiency of the promoter. Moreover, the efficiency of promoters may be increased by altering their sequences, or they may be completely replaced with more efficient promoters, even in organisms of different genera.

[0182] Create functional polypeptide variants Furthermore, a person skilled in the art can determine that sequence identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% is achievable for any of sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75. They are familiar with methods for generating functional variants encoded by nucleic acid molecules that contain a nucleotide sequence encoding a polypeptide, and / or a nucleotide sequence having at least 70% sequence identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or their reverse complements.

[0183] Depending on the technique used, those skilled in the art can introduce completely random or more targeted mutations into genes or non-coding nucleic acid regions (e.g., those important for modifying expression) and subsequently generate a gene library. The molecular biological methods necessary for this purpose are known to those skilled in the art and are described, for example, in Sambrook and Russell (Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001).

[0184] Methods for modifying genes, and therefore methods for modifying polypeptides encoded by genes, have long been known to those skilled in the art, for example, as follows: - Site-directed mutagenesis in which individual nucleotides or several nucleotides of a gene are substituted in a specified manner (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey). - Saturated mutagenesis that allows the exchange or addition of any amino acid codon at any location in a gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22:1593; Barettino D, Feigenbutz M, Valcarel R, Stunnenberg HG (1994) Nucleic Acids Res 22:541; Barik S (1995) Mol Biotechnol 3:1), - Error-prone DNA polymerase chain reaction, which involves mutation of nucleotide sequences by error-prone DNA polymerase (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18:3739); - SeSaM method (sequence saturation method) that inhibits desirable conversion by polymerase, Schenk et al. (Biospektrum, Vol. 3, 2006, 277-279) - For example, a defect in the DNA repair mechanism increases the rate of nucleotide sequence mutations, or the passage of genes in mutagenic strains (Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E. coli mutator strain. In: Trower MK (Ed.) In vitro mutagenesis protocols. Humana Press, New Jersey), or DNA shuffling (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747) involves forming and digesting a pool of closely related genes, and then using these fragments as templates for polymerase chain reactions that ultimately generate full-length mosaic genes through repeat strand separation and recombination.

[0185] Using so-called directed evolution (in particular, as described in Reetz MT and Jaeger KE (1999), Topics Curr Chem 200:31; Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods for optimizing industrial polypeptides by directed evolution, In: Demain AL, Davies JE (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), those skilled in the art can produce functional variants on a large scale in a directed manner. For this purpose, in the first step, a gene library of each polypeptide is first generated, for example, using the aforementioned method. The gene library is then expressed in a suitable manner, for example, by bacteria or by a phage display system.

[0186] The relevant genes of a host organism expressing a functional mutant with characteristics that adequately correspond to the desired traits may follow other mutation cycles. The mutation and selection or screening steps may be repeated until the existing functional mutants possess the desired traits to a sufficient degree. Using this iterative procedure, a limited number of mutations, e.g., 1, 2, 3, 4, or 5 mutations, may be performed stepwise and evaluated and selected for their effects on the activity in question. The selected mutants are then sent to further mutation steps in the same manner. This significantly reduces the number of individual mutants that need to be investigated.

[0187] The results obtained according to the present invention also provide important information regarding the structure and sequence of the relevant polypeptides, which is necessary for generating further polypeptides with desired modified properties in a targeted manner. In particular, it is possible to define so-called "hot spots," i.e., sequence segments that are potentially suitable for modifying properties by introducing targeted mutations.

[0188] Information regarding the position of amino acid sequences can also be estimated, and mutations that likely have little effect on activity may occur in those regions, potentially designing them as "silent mutations."

[0189] Constructs for generating polypeptides of the present invention The nucleotide sequence may be part of an expression cassette. The terms expression cassette and expression construct are used synonymously. Preferably, the recombinant expression construct comprises a nucleotide sequence encoding the polypeptide according to the present invention and under the genetic control of a regulatory nucleic acid sequence.

[0190] In the process applied according to the present invention, the expression cassette may be part of an expression vector, particularly a recombinant expression vector.

[0191] In accordance with the present invention, "expression unit" is understood to mean a nucleic acid having expression activity that modulates expression after functionally binding to a nucleic acid or gene to be expressed, i.e., transcription and translation of the nucleic acid or gene, and including a promoter as defined herein. Therefore, in this context, it is also referred to as a "regulatory nucleic acid sequence." In addition to the promoter, other regulatory elements, such as enhancers, may also be present.

[0192] The terms "expression cassette" or "expression construct" are understood to mean an expression unit functionally linked to a nucleic acid or gene to be expressed in accordance with the present invention. Therefore, in contrast to an expression unit, an expression cassette includes not only nucleic acid sequences that regulate transcription and translation, but also nucleic acid sequences that are to be expressed as proteins as a result of transcription and translation.

[0193] In the context of this invention, the terms “expression” or “overexpression” refer to the generation or increase of the intracellular activity of one or more polypeptides in a microorganism encoded by the corresponding DNA. For this purpose, for example, it is possible to introduce a gene into an organism, replace an existing gene with another gene, increase the copy number of a gene, use a strong promoter, or use a gene encoding a corresponding polypeptide with high activity, and optionally, these measurements can be combined.

[0194] Preferably, such a construct according to the present invention includes a 5'-upstream promoter and a 3'-downstream terminator sequence of each code sequence, and optionally other conventional regulatory elements, each functionally linked to the code sequence.

[0195] "Promoter," "nucleic acid having promoter activity," or "promoter sequence" are understood to mean a nucleic acid that, when functionally linked to a nucleic acid to be transcribed according to the present invention, regulates the transcription of the said nucleic acid.

[0196] In this description, “functional” or “operational” linkage is understood to mean, for example, a contiguous arrangement of one nucleic acid having promoter activity, one nucleic acid sequence to be transcribed, and optionally further regulatory elements, such as nucleic acid sequences that ensure the transcription of the nucleic acid, e.g., terminators, in a manner that allows each regulatory element to perform its function during transcription of the nucleic acid sequence. This does not necessarily require direct linkage in a chemical sense. Gene regulatory sequences, such as enhancer sequences, can exert their function on the target sequence from a more distant location or from other DNA molecules. A preferred arrangement is one in which the nucleic acid sequence to be transcribed is located after the promoter sequence (i.e., at the 3' end) and the two sequences are covalently linked together. The distance between the promoter sequence and the nucleic acid sequence to be expressed by recombination may be less than 200 base pairs, less than 100 base pairs, or less than 50 base pairs.

[0197] In addition to promoters and terminators, other regulatory elements may include: target sequences, enhancers, polyadenylation signals, selection markers, amplification signals, and origins of replication. Suitable regulatory sequences are described, for example, in Goeddel (Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)).

[0198] The nucleic acid constructs according to the present invention particularly include sequences encoding polypeptides derived from, for example, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 or their reverse complements, or derivatives and homologs thereof, and nucleic acid sequences that may be derived therefrom and are operationally or functionally linked to one or more regulatory signals to advantageously control, for example, increase gene expression.

[0199] In addition to these regulatory sequences, the innate regulation of these sequences may still exist before the actual structural gene and may be optionally genetically modified to turn off the innate regulation and enhance gene expression. However, nucleic acid constructs may also be simpler structures, i.e., structures in which no additional regulatory signals are inserted before the coding sequence and the innate promoter with its regulation is not removed. Instead, the innate regulatory sequences may be mutated so that regulation does not occur and gene expression is increased.

[0200] A preferred nucleic acid construct also advantageously includes one or more of the aforementioned “enhancer” sequences functionally linked to the promoter, which enable enhanced expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, may be inserted at the 3' end of the DNA sequence. One or more copies of the nucleic acid according to the present invention may be present in the construct. In the construct, other markers, such as genes complementing nutritional requirements or antibiotic resistance, may optionally be present for construct selection.

[0201] Examples of suitable regulatory sequences include promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, and lacI. q , T7, T5, T3, gal, trc, ara, rhaP(rhaP BAD )SP6, Lambda P R Or Lambda P L These are present in the promoters and are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are present, for example, in the Gram-positive promoters amy and SpO2, and in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, and ADH. Artificial promoters can also be used for regulation.

[0202] For expression in a host organism, nucleic acid constructs are favorably inserted into vectors, such as plasmids or phages, thereby enabling optimal gene expression in the host. The term "vector" can also be understood to mean, in addition to plasmids and phages, all other vectors known to those skilled in the art, namely, viruses, such as SV40, CMV, baculoviruses and adenoviruses, transposons, IS elements, phasmids, cosmids and linear or circular DNA. These vectors can be automatically replicated within a host organism or on chromosomes. These vectors represent a further development of the present invention.

[0203] Suitable plasmids include, for example, E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, and pIN-III. 113 -B1, λgt11 or pBdCI, pIJ101, pIJ364, pIJ702 or pIJ361 of the genus Streptomyces, pUB110, pC194 or pBD214 of the genus Bacillus, pSA77 or pAJ667 of the genus Corynebacterium, pALS1, pIL2 or pBB116 of fungi, 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 of yeast, or pLGV23 or pGHlac of plants + These plasmids are pBIN19, pAK2004, or pDH51. The aforementioned plasmids are a small selection of possible plasmids. Further plasmids are well known to those skilled in the art and can be found, for example, in the book on cloning vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0204] In further development of vectors, nucleic acid constructs or vectors containing nucleic acids according to the present invention may be advantageously introduced into microorganisms in the form of linear DNA and incorporated into the genome of the host organism via heterologous or homologous recombination. This linear DNA may consist of a linearized vector, such as a plasmid, or solely of nucleic acid constructs or nucleic acids according to the present invention.

[0205] For optimal expression of heterologous genes in organisms, it is advantageous to modify nucleic acid sequences to match specific "codon usage" used in the organism. "Codon usage" can be readily determined by computer evaluation of other known genes in the organism in question.

[0206] The expression cassette according to the present invention is produced by fusing a suitable promoter to a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Conventional recombination and cloning techniques are used for this purpose and are described, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0207] For expression in a suitable host organism, recombinant nucleic acid constructs or gene constructs are favorably inserted into host-specific vectors that enable optimal gene expression in the host. Vectors are well known to those skilled in the art and can be found, for example, in "cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0208] The content of documents being cross-referenced shall be considered incorporated by reference.

[0209] The nucleic acid and amino acid sequences identified and / or used herein are listed below: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0210] The following examples are illustrative and are not intended to limit the scope of the invention as set forth in the abstract, specification, or claims.

[0211] A number of possible modifications, which will become immediately apparent to those skilled in the art after considering the disclosures provided herein, also fall within the scope of the present invention.

[0212] All materials and microorganisms or cells used are commercially available products.

[0213] Unless otherwise specified, recombinant proteins should be processed using standard methods, e.g., Sambrook, J., Fritsch, EF and Maniatis, T., Molecular cloning: A Laboratory Manual, 2 ndCloning and expression are performed using the method described in Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0214] Examples Example 1: Identification and functional characterization of mouse and human odor receptors activated by geonol, DMTS, 1-octen-3-ol, butyric acid, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, and transpyrrole. Novel odor receptors were identified according to the method disclosed in International Publication No. 2014 / 210585. Simply put, mouse olfactory neurons were exposed to the following MCS: geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole, and Ca 2+Screening was performed using imaging techniques. Neurons activated by DMTS were further isolated for whole transcriptome analysis to identify sensitive odor receptors. cDNA corresponding to the isolated cellular mRNA was generated and amplified using a PCR-based method (Clontech / Takara, SMARTer® Ultra® Low Input RNA Kit for Sequencing - v3, cat. 634848). The amplified cDNA was then used to generate an Illumina cDNA library and 100-base pair single read sequences for next-generation sequencing. The sequences were aligned to a mouse-related genome (e.g., UCSC version mm10) to generate a complete transcriptome. Since a single odor receptor (OR) was strongly transcribed for olfactory neurons, subsequent identification of MCS-responsive ORs could be achieved. Corresponding human ORs were then identified using phylogenetic relationship assessment with sequence similarity search. The following ORs were identified: Olfr263, Olfr96, Olfr1487, Olfr339, Olfr641, Olfr1126, Olfr46, Olfr1322, Olfr93, Olfr398, Olfr120, Olfr1364, and Olfr937.

[0215] In the second method, a panel of odor receptors containing a repertoire of 400 putative functional human receptors was transfected into cultured human HEK293T cell lines described in International Publication No. 2014 / 210585, expressing the human olfactory G protein alpha subunit (Golf-SEQ ID NO: 49), and conjugated to a cyclicAMP reporter. The panel of odor receptors was exposed to MCS in vitro, and the level of cAMP signaling was measured for each receptor. Subsequently, receptors showing signals significantly different from the baseline were tested in functional dose-response experiments. With respect to Figure 1, the following human odor receptors were identified: OR11A1, OR2M3, OR4S2, OR2J3, OR4Q3, OR51E1, OR2W1, OR1A1, OR5K1, and OR51I2.

[0216] Functional dose-response experiments were performed to evaluate the level of MCS-induced activity in modified cell lines expressing individual putative odor receptors using geonol, DMTS, 1-octen-3-ol, butyrate, 3-methyl-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, or transpyrrole, according to the assays and methods disclosed in International Publication No. 2014 / 210585.

[0217] Example 2: Identification of butyrate inhibitors of olfactory receptors Cell lines expressing the OR51E1 olfactory receptor were used as an antagonist screening platform to identify compounds that reduce the activity of butyrate-induced receptors. These cell lines were then screened in a volatile compound library for their inhibitory properties and potential butyrate odor inhibition. Initially, individual two-component mixtures of butyrate and each test compound were presented to the cells.

[0218] Monitoring single concentrations of butyrate-induced cell activity in the presence or absence of the test compound allowed for the identification of compounds with estimated inhibitory or suppressive effects. These hits were further confirmed by inhibitory dose-response assays to evaluate the inhibition rate (%) normalized to baseline agonist activity.

[0219] The dose-dependent decrease in receptor activity is due to butyrate (EC2). 87 The increasing concentrations of the test compound were recorded in the presence of a single active concentration of ), and the corresponding dose-response inhibition curves were obtained. A summary of the maximum inhibition rate (%) for each tested compound is shown in Figure 2.

[0220] An assay system was designed to select highly selective antagonists, and data were obtained using high concentrations of butyrate (EC87). These data demonstrate that the compound of formula (I) is an olfactory receptor antagonist of butyrate. However, some compounds acted better than others, as suggested by the relative magnitude of the inhibition of olfactory receptors by butyrate.

[0221] Example 3: Identification of olfactory receptor inhibitors of Geonol Cell-based screening: Cell lines expressing OR11A1 or OR2M3 olfactory receptors were used as antagonist screening platforms to identify compounds that reduce the activity of geonol-induced receptors. Each cell line was screened in a volatile compound library for their inhibitory properties and potential geonol-induced odor inhibition. Initially, individual two-component mixtures of geonol and each test compound were presented to the cells.

[0222] Single-point monitoring of geonol-induced cell activity in the presence or absence of the test compound allowed for the identification of compounds with putative inhibitory or deterrent effects. These hits were further confirmed by inhibitory dose-response curve assays. 50 The potency of inhibition was evaluated as a measured value (the inhibitor concentration at which receptor activity is inhibited by half the maximum inhibitory effect level of a given test compound).

[0223] The dose-dependent decrease in receptor activity was observed in Geonol (EC). 80 The increasing concentrations of the test compounds were recorded in the presence of a single active concentration of ), and the corresponding dose-response inhibition curves were obtained. The compounds listed in Table 2 are examples of compounds that reduced the geonol-induced activity of at least one olfactory receptor.

[0224] Sensory evaluation: Panelists were presented with five 16oz glass vials containing either REF (Geonol only), a control (Geonol only), an antagonist only, a mixture of Geonol and an isoconcentration antagonist, or a mixture of Geonol and a low-concentration antagonist (the last four samples were randomized among the panelists). Panelists were first asked to acclimate to the quality of the soil / moss odor from the REF jar, and then to rate the pleasantness, soil / moss odor intensity, and overall odor intensity of the four samples on a linear scale from 0 to 10. The interval between stimuli between samples was 2 minutes to avoid adaptation. The results are shown in Figure 4, which shows the percentage of remaining Geonol malodorous odor (soil / moss odor) in the two-component isoconcentration mixtures of Geonol and 25 different antagonists relative to the REF samples.

[0225] One-way analysis of variance (ANOVA) with Duncan's mean comparison at the 95% confidence level was performed to compare the mean soil / moss odor intensities of the samples of geonol only, the samples of antagonist only, and the geonol standard / antagonist mixtures at two different ratios. The results of the cell-based screening and sensory evaluation are shown in Table 2 and Figure 4.

[0226] * means that no significant difference in soil / moss odor intensity was found between the equi-intensity mixture and the antagonist, and it was significantly lower than the sample of geonol only at the 95% confidence level.

[0227] * means that the soil / moss odor intensity was significantly higher in the equi-intensity mixture than in the sample of antagonist only, and it was significantly lower than the sample of geonol only at the 95% confidence level.

[0228] NS means that no significant difference in soil / moss odor intensity was found between the equi-intensity mixture and the antagonist, and no significant difference was found between the equi-intensity mixture and the sample of geonol only at the 95% confidence level.

[0229] NS means that the soil / moss odor intensity was significantly higher in the equi-intensity mixture than in the sample of antagonist only, and no significant difference was found between the equi-intensity mixture and the sample of geonol only at the 95% confidence level.

[0230]

Table 2-1

Table 2-2

Table 2-3

[0231] Example 4: Identification of an Inhibitor of the Olfactory Receptor for 1-Octen-3-ol Cell lines expressing OR2W1 or OR1A1 olfactory receptors were used as antagonist screening platforms to identify compounds that reduce the activity of 1-octen-3-ol induced receptors. Each cell line was screened in a volatile compound library for their inhibitory properties and potential 1-octen-3-ol odor inhibition. Initially, individual two-component mixtures of 1-octen-3-ol and each test compound were presented to the cells.

[0232] Monitoring single concentrations of 1-octen-3-ol-induced cell activity in the presence or absence of the test compound allowed for the identification of compounds with putative inhibitory or deterrent effects. These hits were further confirmed by inhibitory dose-response assays. 50 Potency and efficacy of inhibitory activity were evaluated as measured values ​​(inhibitor concentration at which receptor activity is inhibited by half the maximum inhibitory effect level of a given test compound) and therefore as inhibition rate (%) (normalized to baseline agonist activity).

[0233] The dose-dependent decrease in receptor activity was observed in 1-octen-3-ol (EC) 80 The increasing concentrations of the test compounds were recorded in the presence of a single active concentration of ), and the corresponding dose-response inhibition curves were obtained. The compounds listed in Table 3 are examples of compounds that reduced the 1-octen-3-ol induced activity of at least one receptor.

[0234] [Table 3-1] [Table 3-2]

[0235] Example 5: Identification of inhibitors of butyric acid odor receptors A cell line expressing the OR51E1 olfactory receptor was used as an antagonist screening platform to identify compounds with the property of reducing the activity of butyric acid-induced receptors. The stable cell line was screened with a volatile compound library for their inhibitory properties and potential odor inhibition of butyric acid. First, individual binary mixtures of butyric acid and each test compound were presented to the cells.

[0236] Monitoring a single concentration of butyric acid-induced cell activity in the presence or absence of the test compound enabled the identification of compounds with an estimated inhibitory or blocking effect. These hits were further confirmed in an inhibitory dose-response assay to evaluate the potency of activity inhibition as the IC 50 measurement (the inhibitor concentration at which receptor activity is inhibited by the level of half-maximal inhibitory effect of a given test compound).

[0237] The dose-dependent decrease in receptor activity was recorded by increasing concentrations of the test compound in the presence of a single active concentration of butyric acid (EC 94 ), and the corresponding dose-response inhibition curve was obtained. The compounds listed in Table 4 are examples of compounds that decreased the butyric acid-induced activity of at least one receptor.

[0238]

Table 4-1

Table 4-2

Table 4-3

[0239]

Table 5

[0240] Example 6: Identification of Inhibitors of the Olfactory Receptor of DMTS Cell lines expressing the odor receptors OR52N5, OR2L13, OR2AJ1, OR4C15, OR5AC2, OR8H3, OR11G2, OR52N2, or OR5T1 were used as antagonist screening platforms to identify compounds that reduce DMTS-induced receptor activity according to the assays and methods disclosed in International Publication No. 2014 / 210585. Each cell line expressing the human odor receptor was screened in a volatile compound library for their inhibitory properties and potential DMTS odor inhibition. Initially, individual two-component mixtures of DMTS and each test compound were presented to the cells. Single-point monitoring of DMTS-induced cell activity in the presence or absence of the test compound allowed for the identification of compounds with putative inhibitory or suppressive effects. These hits were further confirmed by inhibitory dose-response curve assays for IC 50 The potency of inhibitory activity was evaluated as the measured value (inhibitor concentration at which receptor activity is inhibited by half the maximum inhibitory effect level of a given test compound). The dose-dependent decrease in receptor activity was defined as DMTS (EC). 80 The increasing concentrations of the test compounds were recorded in the presence of a single active concentration of ), and the corresponding dose-response inhibition curves were obtained. The compounds listed in Table 5 are examples of compounds that reduced the DMTS-induced activity of at least one receptor.

[0241] [Table 6]

Claims

1. A method for identifying compounds that bind to olfactory receptors activated by substances that cause laundry odors, and that suppress, block, inhibit, and / or modulate the activity of said olfactory receptors, a. Contacting an olfactory receptor polypeptide that is OR2M3; or an olfactory receptor polypeptide that has at least 90% amino acid sequence identity with an olfactory receptor polypeptide that is OR2M3 and is activated by Geonol, with the test substance and the substance that causes the malodorous odor of the laundry; and b. Measuring the response of the olfactory receptors to substances that cause laundry odors by measuring the response of the olfactory receptors in the presence and absence of the test substance. The method, wherein the substance that causes the malodor of the laundry includes Geonol.

2. moreover, c. Identifying test substances that modulate the response of the olfactory receptors based on responses measured in the presence and absence of the test substance, and d. Select a test substance identified as a compound that modulates the response of the olfactory receptors to substances that cause laundry odors. The method according to claim 1, including the method described in claim 1.

3. The olfactory receptor polypeptide is a. Containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 14; and / or b. Encoded by a nucleic acid molecule containing a nucleotide sequence having at least 90% sequence identity with respect to Sequence ID No. 13 or its reverse complement, The method according to claim 1 or 2.

4. The olfactory receptor polypeptide is a. Containing an amino acid sequence having at least 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14; and / or b. Encoded by a nucleic acid molecule containing a nucleotide sequence having at least 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13 or its reverse complement, The method according to claim 3.

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