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WO2008017598A2 - Récepteurs de la substance odorante citrusal - Google Patents

Récepteurs de la substance odorante citrusal Download PDF

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Publication number
WO2008017598A2
WO2008017598A2 PCT/EP2007/057801 EP2007057801W WO2008017598A2 WO 2008017598 A2 WO2008017598 A2 WO 2008017598A2 EP 2007057801 W EP2007057801 W EP 2007057801W WO 2008017598 A2 WO2008017598 A2 WO 2008017598A2
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WO
WIPO (PCT)
Prior art keywords
receptor
fragrance
formula
or7a5
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2007/057801
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German (de)
English (en)
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WO2008017598A3 (fr
Inventor
Hanns Hatt
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Symrise AG
Original Assignee
Symrise AG
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Filing date
Publication date
Application filed by Symrise AG filed Critical Symrise AG
Publication of WO2008017598A2 publication Critical patent/WO2008017598A2/fr
Publication of WO2008017598A3 publication Critical patent/WO2008017598A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/11—Aldehydes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • the invention relates to the field of olfactory receptors, the medication affecting the condition and the biosensors.
  • fragrance receptor gene For example, seven years elapsed between the first functional description of a fragrance receptor gene and its corresponding fragrance ligand (Zhao et al., Functional expression of a mammalian odorant receptor, Science 279 (1998), 237-242). Until now, only two human fragrance receptor genes have been able to associate corresponding fragrances with fragrance receptors:
  • fragrance receptor genes in heterologous systems has not been reliably achieved despite constant efforts of the experts (Mombaerts, loc. Cit.). Furthermore, it is still not possible to make predictions about which substances can act as agonists or antagonists on the basis of the DNA sequence or the receptor amino acid sequence. For example, findings are based on Can hardly be transferred to other species because sperm cells are susceptible to random gene expression and fragrance receptor genes found in sperm can be difficult to associate with the remaining fragrance receptor gene families (Vanderhaeghen et al, Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species; Genomics 39 (1997), 239-246).
  • an expression system comprising a cell expressing an OR7A5 fragrance receptor or fragrance receptor having an amino acid sequence homology of at least 70% to the amino acid sequence of the transmembrane domains III to VI of the OR7A5 fragrance receptor
  • the fragrance receptor is heterologous with respect to the cell or
  • the starting point here was the nucleic acid sequence published under the name OR7A5 (EMBL accession number BC104809, see Strausberg et al., Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences; Proc. Natl. Acad 99 (26): 16899-16903 (2002)) of the receptor-encoding gene (gene sequence) of a human perfume receptor.
  • OR7A5 EBL accession number BC104809, see Strausberg et al., Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences; Proc. Natl. Acad 99 (26): 16899-16903 (2002)
  • OR19-17 for nomenclature see Ben-Arie et al., Human Molecular Genetics (1994), 229-235).
  • the receptor belongs to the class of proteins described at the outset, which belong to the superclass of G protein-coupled proteins with seven putative transmembrane domains.
  • a fusion protein according to the invention is also a fragrance receptor having an amino acid sequence homology of at least 70% to the amino acid sequence of the transmembrane domains III to VI of the OR7A5 fragrance receptor.
  • Transmembrane domain III begins at amino acid 99 and transmembrane domain VI ends at amino acid 259 of the complete OR7A5 protein.
  • Particular preference is given to those proteins which have a protein segment having an amino acid sequence homology of more than 70%, preferably 90% and particularly preferably 5, 4, 3, 2 or 1 amino acids, difference to a protein segment consisting of the transmembrane domains III to VI of the OR7A5 fragrance formulation.
  • the amino acid sequence homology may conveniently be calculated using the EMBOSS: water program (Algorithm of Smith and Waterman (1981), Identification of common molecular subsequences, J. Mol. Biol. 147: 195-197) (gap open penalty: 10.0 Gap extension penalty: 0.5, Blosum62 matrix).
  • the program calculates a similarity percentage, which is the measure of homology.
  • a "similarity" percentage value of 80% therefore means a sequence homology of 80% for the purposes of this invention.
  • a fusion protein is additionally indicated a) comprising the transmembrane domains III to VI of the OR7A5 fragrance receptor or a protein segment having an amino acid sequence homology of more than 70%, preferably 90% and particularly preferably 5, 4, 3, 2 or 1 amino acids difference to the protein segment consisting of the transmembrane domains III to VI of the OR7A5 fragrance receptor.
  • fusion protein according to the invention facilitates the production of an expression system according to the invention.
  • this description refers to a receptor or receptor according to the invention, this is the OR7A5 receptor, a fragrance receptor having an amino acid sequence homology of at least 70% to the amino acid sequence of the transmembrane domains III to VI of the OR7A5 fragrance receptor or a fusion protein according to the invention of the type described above unless otherwise clearly stated in the context of the text.
  • the receptor is expressed as a heterologous protein in a host cell - also below more - it is possible that the transcription and / or translation of certain nucleic acid codons or codon sequences proceeds more efficiently than the transcription and / or translation of other codons or codon sequences. This may result in a preference for certain amino acids and / or amino acid sequences. It is now preferred that some or all of the amino acids of the receptor, for which there is no preference of the host cell, are replaced by those amino acids for which there is a preference of the host cell. Further, it may be advantageous to remove a protease cleavage site by an amino acid substitution, deletion or insertion.
  • the receptor is a fusion protein further comprising one or more further protein sections.
  • these may be, in particular, signal peptides such as the "5HT 3 sequence” (cf. CH Wetzel et al., Journal of Neuroscience 19, 7426-7433 (1999)), which facilitates transport and functional incorporation into the cell membrane.
  • signal peptides such as the "5HT 3 sequence” (cf. CH Wetzel et al., Journal of Neuroscience 19, 7426-7433 (1999)), which facilitates transport and functional incorporation into the cell membrane.
  • other signal peptides may also be used in particular those which control or facilitate an intra- and / or intercellular transport of the fusion protein.
  • R 1 and R 2 independently of one another denote H or CH 3 ,
  • X is H, CH 3 , OCH 3 or OC 2 H 5 and
  • the group -C (O) X is bonded to one of the carbon atoms which is linked to the radical R 1 or R 2 .
  • a fusion protein according to the invention is a receptor which is suitable for binding specifically to a compound of formula (I).
  • the receptor binds a substance or specifically binds a substance to the receptor if, when applied to several molecules of the receptor, the substance is at low concentrations, preferably at concentrations less than 1 mM, more preferably at concentrations less than or equal to 1 mM, in particular preferred at Concentrations less than or equal to 100 ⁇ M, most likely binds again and again in the same area of the receptor.
  • the result of such a specific binding may be a - preferably reversible - conformational change of the receptor which is not substantially limited only to the site of binding.
  • an agonist in the context of the invention is understood as meaning a substance which, by virtue of its specific binding to the receptor, triggers a chemical reaction which exceeds this mere bond.
  • receptors are not chemically abrupt next to other cell constituents, but are in operative association with other parts of at least one signal transduction cascade, such as that mediated by G proteins.
  • Specific binding of an agonist converts such receptors from an inactive to an active state, usually accompanied by a conformational change of the receptor that is not substantially limited to the site of agonist binding, and is manifested in turn on of the signal transduction cascade, recognizable, for example an increase in the cytosolic concentration of Ca 2+ , cAMP, cGMP and / or inositol triphosphate or the change in the ATP / ADP ratio.
  • the conformational change of the receptor, and also the turning on of the signal transduction cascade is a chemical reaction beyond the mere binding of the agonist.
  • agonists based on the receptor are, in particular, the compounds of the formula (I).
  • an antagonist is a substance which, although specifically bound by a receptor, makes the specific binding of an agonist to the receptor more difficult.
  • the specific binding of the antagonist unlike an agonist, does not lead to the activation of one
  • Signal transduction cascade when the receptor is part of such by the specific binding of an agonist switched signal transduction cascade.
  • one may be by the specific binding of an agonist caused conformational change of the receptor in the specific binding of an antagonist.
  • the compounds of the formula (I) can be obtained via a Diels-Alder reaction of a 1,3-diene of the formula (II) and an ⁇ , ⁇ -unsaturated carbonyl compound of the formula (III), such as the following reaction scheme to clarify:
  • the starting material of the formula (II) is preferably myrcene (7-methyl-3-methylene-1,6-octadiene) (the dashed line in the formulas (I) and (II) then being a double bond).
  • the compounds of the formula C, D and F (ie those for which the dotted line represents a single bond in formula (I)) can be prepared, for example, by partial hydrogenation from the compounds of the formula A, B and E (ie those for which Formula (I) the dashed line represents a double bond) are synthesized.
  • compounds of the formulas A1 and A2 or B1 and B2 can be converted into compounds of the formulas C1 and C2 or D1 and D2 by means of partial hydrogenation (if appropriate using a protective group for the carbonyl function, for example an acetalization or ketalization) (see also US Pat Angew Chem. 2000, 1 12, 3106-3188).
  • the compounds of the formula (I), in particular the compounds of the formulas A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 and F2 and in particular those mentioned above as newly designated substances of the formula (I) are given according to the invention as fragrances. They are suitable for specific binding to a receptor as described above, in particular to a fusion protein according to the invention and for triggering a signal transduction cascade as described above. It has also been found, as explained in more detail below, that compounds of the formula (I) can increase the Ca 2+ concentration in a cell expressing the receptor described and increase the flagellum frequency of a sperm.
  • the invention therefore also provides for the use of a compound of the formula (I), in particular of the compounds of the formulas A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 and F2 and, in particular, the substances described above as new of formula (I)
  • Fragrance receptor having an amino acid sequence homology of at least 70% to the amino acid sequence of the transmembrane domains III to VI of the OR7A5 fragrance receptor or expressed a fusion protein according to the invention
  • a vector comprising a nucleic acid portion encoding a receptor according to any one of the previous aspects of the invention, that is, a vector comprising a nucleic acid portion encoding a) an OR7A5 fragrance receptor or b) a Fragrance receptor having an amino acid sequence homology of at least 70% to the amino acid sequence of transmembrane domains III to VI of the OR7A5 fragrance receptor or c) a fusion protein of the invention.
  • a vector makes it easier to clone a nucleic acid encoding the receptor of the invention.
  • the vector may also be a so-called suicide vector, particularly one which allows for the integration of the receptor-encoding nucleic acid portion into the genome of the host cell.
  • the vector is an expression vector.
  • Suitable host cells are in particular those from Drosophila melanogaster, Caenorhabditis elegans, Xenopus / aews oocytes and human HEK293 cells as well as CHO (Chinese Hamster Ovary), COS (African Green Monkey Kidney), BHK (Syrian Baby Hamster Kidney) and PC12 - (Rat adrenal phenochromocytoma) cells, with HEK293 cells being particularly preferred.
  • the expression system according to the invention comprises, as indicated above, a cell which overexpresses a receptor according to the invention.
  • "overexpressing” means any process in which the receptor is present beyond the maximum achievable gene expression level for the respective host cell under natural conditions.
  • the receptor may in particular also be a heterologous receptor for the host cell. If the host cell concerned does not normally produce a receptor according to the invention, overexpression in the meaning of the invention already takes place when it (for the first time) ever produces such a receptor.
  • those host cells which, in addition to the functional expression of the receptor, also provide the elements necessary for a signal transduction cascade influenced by the receptor through the binding of an agonist.
  • the host cell is a mammalian cell, wherein the host cell may in particular also be a degenerate mammalian cell.
  • a mammalian cell wherein the host cell may in particular also be a degenerate mammalian cell.
  • An example of this is the human embryonic kidney cell-derived cell line HEK293 (see Graham et al., J. Gen. Virol. (1977): 59-72).
  • a complex is specified, wherein the complex comprises a fusion protein according to the invention, as well as specifically bound thereto, a compound of the formula (I), in particular of the formula A1, A2, B1, B2, C1, C2, D1, D2 , E1, E2, F1 or F2.
  • Such a complex is particularly advantageous for studying the interactions between the receptor and an agonist and / or a possible antagonist.
  • the antagonist may be reversible or irreversible.
  • the receptor When the receptor is in operative association with a signal transduction cascade, the production of such a complex of receptor and agonist enables the activation or activation of the corresponding signal transduction cascade.
  • the production of a receptor-antagonist complex makes it possible to prevent the activation of the signal transduction cascade even in the presence of the agonist.
  • a complex of receptor and agonist or antagonist are described below.
  • a biosensor comprising
  • the biosensor comprises a cell expressing a receptor according to the invention, ie a) an OR7A5 fragrance receptor or b) a fragrance receptor having an amino acid sequence homology of at least 70% to the amino acid sequence of the transmembrane domains III to VI of the OR7A5 fragrance receptor .or or c) expresses a fusion protein according to the invention, and in which the receptor is in active connection with a signal transduction cascade.
  • a receptor-agonist complex is prepared as just described. This may result in a not limited essentially only to the site of binding of the agonist limited conformational change of the receptor.
  • the receptor triggers the signal transduction cascade after binding of the agonist. This in turn generates a measurable signal, for example in the form of an increase in the cytosolic Ca 2+ concentration.
  • a second messenger such as cAMP, cGMP, IP 3 and the like.
  • One skilled in the art can readily select a suitable substance for detecting this binding, the concentration or conformation of which changes according to the specific binding of the agonist to the receptor, based on the signal transduction cascade influenced by the specific binding of an agonist.
  • Such a biosensor is particularly advantageous for finding additional receptors.
  • the skilled person will first Provide an expression system in which the putative receptor is functionally expressed. Suspected agonists are applied to the expression system and an optionally triggered thereby signal of the biosensor is measured. If a substance triggers a biosensor signal and this signal does not also occur in the natural cells of the expression system (ie, cells that have not been changed to an expression system), the expression system will actually express a receptor for that substance.
  • a probe for detecting a nucleic acid encoding a portion of a receptor of the invention is suitably a nucleic acid, in particular an RNA or a, preferably single-stranded, DNA, wherein the nucleic acid can hybridize with a nucleic acid which codes for a portion of a receptor according to the invention.
  • Preferred probes have the sequence SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 or a 12-30, preferably 15-20 amino acids long section of the sequences SEQ ID NO 3 or SEQ ID NO 4 ..
  • the nucleic acid is connected to a detectable label, for example with a fluorescence, luminescence, color coding, a radioactive label or an enzyme that catalyzes the formation of a detectable reaction product.
  • a detectable label for example with a fluorescence, luminescence, color coding, a radioactive label or an enzyme that catalyzes the formation of a detectable reaction product.
  • the probe can also be coupled to a solid, for example to metal particles such as magnetic beads (optionally via a linker).
  • the invention teaches to use a nucleic acid, preferably a probe as previously described, to detect a nucleic acid encoding a fragment of a receptor.
  • a nucleic acid preferably a probe as previously described
  • further receptors with the same or similar amino acid sequence as the receptor according to the invention can be found.
  • the probe is contacted under preferably stringent hybridization conditions with nucleic acids of a cell to be assayed to allow hybridization of the probe to a corresponding host nucleic acid, if any corresponding nucleic acid is present.
  • the less stringent the hybridization conditions the less similar can be the nucleic acids that hybridize to the probe.
  • the nucleic acids hybridized with the probe are purified and sequenced.
  • nucleic acids coding for a fragment of a receptor it is sufficient to bring these nucleic acids into contact with the probe, preferably under stringent hybridization conditions, in order to hybridize the probe to the corresponding probe
  • the nucleic acid to be detected by the probe is immobilized on a solid support, for example in a gel or on a nylon filter.
  • hybridizing the probe to the nucleic acid to be detected is readily detectable by also binding the probe - mediated by the nucleic acid to be detected - to the solid support and substantially not washing off the solid support when performing a wash step.
  • Those skilled in such detection methods are known as Southern or Northern Blot.
  • a receptor according to the invention for detecting and / or specifically binding a compound of the formula (I), in particular of the formula A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 or F2.
  • a particularly preferred use of a compound of the formula (I), in particular of the formula A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 or F2, is to influence the flagellum frequency of a sperm. It has been found that even sperm receptors according to the first Aspect of the invention express and chemokinetic reactions that can be triggered by the binding of a compound of formula (I), in particular myracaldehyde.
  • the addition of an antagonist to sperm leads to the effects described - with constant agonist concentration - either not or not to the usual extent.
  • the one or more compound (s) of the formula (I) can be present in particular as a pharmaceutically acceptable salt of the particular compound of the formula (I).
  • a drug is understood to be any agent used for the prevention, diagnosis or treatment of a physical condition of a human and / or animal.
  • Medicaments in the sense according to the invention can therefore be, in particular, medicaments according to ⁇ 1 of the Medicinal Products Act, but also medical products according to ⁇ 3 of the Medical Devices Act, in their version valid on August 1, 2002.
  • a drug utilizes the benefits achievable with the use of agonists and antagonists described above.
  • the medicament suitably comprises a pharmaceutically acceptable carrier.
  • the medicament may also comprise a precursor of the agonist or antagonist, which is converted into an agonist or antagonist in the human and / or animal body.
  • an antagonist - preferably an antagonist irreversibly inhibiting the receptor - in or on the human body, preferably in the vagina, uterus and / or fallopian tube, there to sperm optionally present there orientation to complicate an ovum that may be present.
  • This can be done by an intravaginal preparation from which the antagonist is released.
  • a condom with an antagonist - again preferably an irreversible -, for example, to impregnate or coat, so that the antagonist is released when used properly the condom.
  • a contraceptive drug it may be particularly advantageous to release one or more agonists - optionally in combination with other substances - in the fallopian tube and / or uterus to accelerate spermatozoa with a low flagellum frequency.
  • an antagonist against the specific binding of an agonist of the formula (I), in particular the A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 or F2, in particular with X H to influence odor perception ability of a human and / or an animal.
  • an antagonist makes it possible in a simple manner to raise the odor perception threshold for one or more agonists in humans and / or animals, so that the agonist concerned is less easily smelled. This may be particularly useful in cases where an agonist develops a disturbing odor, but can only be removed with disproportionate effort from the air or other carrier medium.
  • a method for producing a fusion protein or expression system according to the invention, a method is disclosed which comprises the steps:
  • a method for detecting a compound of the formula (I), in particular the A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 or F2, in particular where X H, in a sample, the method comprising the steps of:
  • the receptor may be provided in the form of an expression system in which the receptor is operatively linked to a signal transduction cascade.
  • a specific binding of the compound of formula (I) to the receptor can be checked by switching on the relevant signal transduction cascade, while the specific binding of the antagonist can be checked by the absence of such switching on the signal transduction cascade in the presence of a compound of formula (I).
  • an expression system can also be simulated artificially by assembling the receptor and the other components of the signal transduction cascade in vitro.
  • the receptor may also be present as a purified protein bound to the surface of a solid support, particularly a metal surface.
  • a physical surface property may change. This may, for example, be the change in surface plasmon resonance, as may be determined in a Biacore process.
  • the receptor and / or a compound of the formula (I) and / or an antagonist with a fluorescent dye and the change brought about by the specific binding of the compound of the formula (I) and / or of the antagonist to the receptor to determine the fluorescence.
  • the receptor may be present as a fusion protein with a green fluorescent p / Ote / n (GFP) portion, so that the fluorescence of the fusion protein or the GFP portion of the fusion protein upon specific binding of a compound of formula (I) and / or an antagonist changes accordingly.
  • FRET assays fluorescence resonance energy transfer assays
  • the receptor may also be present as a fusion protein with a luciferase moiety, such that the luminescence of the fusion protein or luciferase moiety of the fusion protein changes upon specific binding of an agonist and / or an antagonist ("BRET assay").
  • a method for detecting the antagonist in a sample comprising the following steps: a) providing a receptor according to the invention, in particular an OR7A5 receptor, a fragrance receptor having an amino acid sequence homology of at least 70% to the amino acid sequence of the transmembrane domains III to VI of the OR7A5 fragrance receptor or a novel
  • steps b) and c) can be carried out simultaneously or in any order one after the other, provided that step c) is carried out before or simultaneously with step d).
  • step d) is carried out before or simultaneously with step d.
  • the receptor according to the invention is first provided.
  • the person skilled in the art can make use of the possibilities described above for a method according to the invention for detecting an agonist, in particular a compound of the formula (I), and / or antagonists.
  • the receptor is brought into contact with the optionally antagonist-containing sample.
  • conditions are set under which an antagonist could specifically bind to the receptor, if it is present in the sample.
  • the receptor is contacted with an agonist.
  • the conditions are adjusted so that the agonist can specifically bind to the receptor if no antagonist has specifically bound to the receptor.
  • the receptor is first contacted with the agonist and then with the sample optionally containing the antagonist. In this case, it can be concluded from the decrease in the extent to which the agonist is specifically bound to the receptor, the presence of the antagonist and optionally also its concentration in the sample to be examined.
  • Fig. 1 shows the specific activation of HEK293 cells by myracaldehyde.
  • Example 1 Specific activation of HEK293 cells by myracaldehyde
  • HEK293 cells were transiently transfected with a vector encoding OR7A5.
  • the gene for OR7A5 with primers SEQ ID NO. 1 and SEQ ID NO: 2 amplified with the restriction nuclease EcoR V digested and cloned the fragment obtained in the corresponding interface of the vector pCDNA3 (Invitrogen, USA).
  • transfection protocol used in this and the following examples corresponds to that of WO 2004/033496 A1, whose contents, in particular its examples 3 to 13, in so far referenced.
  • HEK293 cells were maintained under standard conditions in DMEM (Sambrook et al., Molecular cloning: A laboratory manual, CoId Spring Harbor 1982) with 10% fetal calf serum, 100 U / ml penicillin and streptomycin, and 2 mM L-glutamine. Transfection was by calcium phosphate precipitation. About 1 h before the start of transfection, the medium was replaced with 2 ml of fresh DMEM. For transfection, 100 to 200 ⁇ l of the transfection reagent were added.
  • FIG. 1 shows a representative illustration of a Ca 2+ distribution in the transfected HEK293 cells.
  • HEK293 cells were transfected with a vector encoding OR7A5. About 5% of the transfected cells showed a significant increase in intracellular calcium content after addition of myracaldehyde.
  • HEK293 cells were transfected as described in Example 1 with vectors encoding OR7A5 or OR1 D2. The transfected cells were examined for their response to various substances.
  • FIG. 2 shows in panel a that the effect of myracaldehyde (“Myrac”) is not reduced by prior administration of n-undecanal, instead There is still a significant increase in the intracellular Ca 2+ content when administering myracaldehyde.
  • Myrac myracaldehyde
  • the responses of sperm to myracaldehyde and was measured (a) by computer-assisted video motion analysis (CAVMA) to detect changes in swimming velocity (sperm activation), and (b) in a flat capillary assay for proximal chemotaxis to examine a source of diffusion material.
  • CAVMA computer-assisted video motion analysis
  • Bioassay data were analyzed statistically by one-way analysis of variance (ANOVA) using a post hoc Scheffe test (R.W. Day and G. P. Quinn, E.col. Monogr. 59, 433-463 (1989)). Bonferroni correction was used to adjust the ⁇ levels for multiple comparisons.
  • Chambers with four separate compartments were used to study sperm chemotaxis, using the capillary method of Adler (Adler, Science 153, 708-716 (1966); Adler, Journal of General Microbiology 74, 77-91 (1984); 1973)). Each compartment consisted of two wells connected by a channel. The sperm samples (10 6 cells / ml) were each presented in the wells. A flat 6 ⁇ L microcapillary tube (Drummond Scientific Co.) containing either test or control solution was introduced into the channel with its two ends contacting the fresh sperm suspensions within the two wells.
  • the spermatozoa responded to myracaldehyde (see Figure 3) in a dose-dependent manner by increasing the speed of swimming
  • FIG. 3 graphically depicts these results, the values averaging and standard deviation being taken from FIGS.
  • Raw data are calculated regression lines. From a concentration of

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Abstract

L'invention concerne le domaine des récepteurs olfactifs, des médicaments influençant la conception et des biocapteurs.
PCT/EP2007/057801 2006-08-11 2007-07-27 Récepteurs de la substance odorante citrusal Ceased WO2008017598A2 (fr)

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Application Number Priority Date Filing Date Title
DE102006037581.5 2006-08-11
DE102006037581A DE102006037581A1 (de) 2006-08-11 2006-08-11 Citrusal-Duftstoffrezeptoren

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WO2008017598A2 true WO2008017598A2 (fr) 2008-02-14
WO2008017598A3 WO2008017598A3 (fr) 2009-01-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9717815B2 (en) 2014-07-30 2017-08-01 Georgia-Pacific Consumer Products Lp Air freshener dispensers, cartridges therefor, systems, and methods

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS55130914A (en) * 1979-03-30 1980-10-11 Hisamitsu Pharmaceut Co Inc Novel antiallergic agent
JPS55133311A (en) * 1979-04-05 1980-10-17 Hisamitsu Pharmaceut Co Inc Novel antiallergic agent
GB9814646D0 (en) * 1998-07-07 1998-09-02 Quest Int Method of reducing or preventing malodour
AU2903701A (en) * 1999-10-08 2001-04-23 Digiscents Olfactory receptor sequences

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9717815B2 (en) 2014-07-30 2017-08-01 Georgia-Pacific Consumer Products Lp Air freshener dispensers, cartridges therefor, systems, and methods
US10391193B2 (en) 2014-07-30 2019-08-27 Gpcp Ip Holdings Llc Air freshener dispensers, cartridges therefor, systems, and methods

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WO2008017598A3 (fr) 2009-01-15

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