WO2008017598A2 - Citrusal aroma receptors and use of myrac aldehyde derivatives for increasing the flagellation frequency of a spermatozoon - Google Patents

Abstract

The invention relates to the field of olfactory receptors, conception-related medication, and biosensors.

Description

 Citrusal perfume receptors

The invention relates to the field of olfactory receptors, the medication affecting the condition and the biosensors.

Since the discovery of the DNA sequences (genes) coding for olfactory receptors (fragrance receptors), numerous aspects related to these receptors are still unclear. Although the skilled person with some certainty on the basis of characteristic features - such as belonging to the family of G-protein coupled receptors, hypervariability in the transmembrane domains III, IV, V and VI - between DNA sequences of fragrance receptors and such sequences, not for a Coding for fragrance receptor. However, this is still not substantively known about the regulation of perfume receptor expression; in particular, it is unknown in which tissue and at what strength a suspected perfume receptor gene is expressed; furthermore, it is unpredictable which substances can specifically bind to the fragrance receptor encoded by this gene, whether as an agonist (activator) or as an antagonist (inhibitor). Although vertebrate fragrance receptor genes can be relatively easily subcloned by degenerate PCR (polymerase chain reaction) (Mombaerts, Genes and ligands for odorant, vomeronasal and taste receptors, Nat Rev. Neurosci 5 (2004), 263-278). However, it has hitherto proven to be very difficult to associate an isolated fragrance receptor with an associated fragrance (ligand) detectable by the receptor. 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:

Wetzel et al (The Journal of Neuroscience, 1999: 7426-7433) describe the functional expression of the human hOR17-40 receptor protein. Only two substances acting as agonists could be determined, namely helional and heliotropylacetone. These substances trigger activation of the receptor, which results in a transient increase in cytosolic Ca ²⁺ levels when the receptor is functionally expressed in HEK293 cells. Antagonists, ie substances that can specifically and reversibly prevent receptor activation by one or more agonists, were not found. Spehr et al (Identification of a testicular odorant receptor mediating human sperm chemotaxis; Science 299 (2003), 2054-2058) again showed the binding of bourgeonal to another human fragrance receptor (hOR17-4) and the antagonistic effect of n-undecanal demonstrate this ligand binding.

However, the functional expression of 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).

There is therefore a great need to find additional fragrance receptors, to express functionally and to determine the respectively effective agonists and antagonists.

According to the invention, therefore, there is provided 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

a) the fragrance receptor is heterologous with respect to the cell or

b) the fragrance receptor is overexpressed in the cell.

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. A synonymous name is 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. Since the functional expression of this receptor has now been achieved for the first time, it has also become possible to find substances that can specifically bind to the receptor as an agonist or antagonist; more below. The skilled artisan will recognize that even a protein having a protein portion with an amino acid sequence other than the sequence of OR7A5 may be a fragrance receptor having the fragrance ligand specificity described below. The chances of obtaining a functional receptor, in particular a fragrance receptor, is higher in the group of proteins with a homologous amino acid sequence to OR7A5 than in the group of proteins without or with little homology to the sequence of this fragrance receptor. Accordingly, 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.

According to the 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. B) the portion according to a) between the transmembrane domains I and VI hOR17-40 (Wetzel et al., Supra) and between the transmembrane domain I from hOR17-40 and the transmembrane domain III according to a) another functional transmembrane domain is arranged. For the production and use of such fusion proteins, the person skilled in the art will be guided in particular by the description of WO 2004-033496A1, the content of which is referred to in so far. In particular, a fusion protein according to the invention facilitates the production of an expression system according to the invention.

As far as 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.

If 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.

Preferably, 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. However, instead of or in addition to the "5HT ₃ " sequence, other signal peptides may also be used in particular those which control or facilitate an intra- and / or intercellular transport of the fusion protein.

It has now been found that the receptor binds specifically to compounds of the following formula (I):

(I)

in which

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 dashed line drawn in the formula (I) means either a single or a double bond, 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 .

Accordingly, a fusion protein according to the invention is a receptor which is suitable for binding specifically to a compound of formula (I).

Under a specific binding in the context of this invention, a bond is understood, which - in simple terms - works according to a kind key-lock principle. In particular, 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 μM, most likely binds again and again in the same area of the receptor. In particular, 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.

Furthermore, 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. In their natural environment, 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. In the context of this invention, agonists based on the receptor are, in particular, the compounds of the formula (I).

For the purposes of the invention, 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. In particular, one may be by the specific binding of an agonist caused conformational change of the receptor in the specific binding of an antagonist.

In particular, binding of the receptor to the compounds of formulas A (comprising A1 and A2), formulas B (comprising B1 and B2), formulas C (comprising C1 and C2), formulas D (comprising D1 and D2), formulas E (comprising E1 and E2) and formulas F (comprising F1 and F2):

A1 A2

B1 B2

C1 C2

D1 D2

E1 E2

and

F1 F2

in which X has the abovementioned meaning.

Preference is given here to the compounds of the formulas B1 and B2 and mixtures thereof, in particular those compounds of the formulas B1 and B2 in which X is H in each case. Particularly preferred is the compound of formula B1 with X = H. New are the following substances:

Compounds of the formula A2 in which X = OC ₂ H ₅ ,

Compounds of the formula B2 in which X = OC ₂ H ₅ ,

Compounds of the formula C1 in which X denotes CH ₃ , OCH ₃ or OC ₂ H ₅ ,

Compounds of the formula C2 in which X is CH ₃ , OCH ₃ or OC ₂ H ₅ ,

Compounds of the formula D1 in which X is CH ₃ or OC ₂ H ₅ ,

Compounds of the formula D2 in which X denotes CH ₃ , OCH ₃ or OC ₂ H ₅ ,

Compounds of the formula E1 in which X is OCH ₃ or OC ₂ H ₅ ,

Compounds of the formula E2 in which X is OCH ₃ or OC ₂ H ₅ ,

Compounds of the formula F1 in which X denotes H, CH ₃ , OCH ₃ or OC ₂ H ₅ ,

Compounds of the formula F2 in which X is H, CH ₃ , OCH ₃ or OC ₂ H ₅ .

Based on US Pat. No. 2,842,598, 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 α, β-unsaturated carbonyl compound of the formula (III), such as the following reaction scheme to clarify:

(H) (Hi) wherein the dashed line, R ¹ , R ² and X are each as defined above.

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).

As starting materials of the formula (III) it is preferred to use acrolein, methacrolein or crotonaldehyde, it also being possible to generate the educts of the formula (III) in situ.

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. For example, 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).

Compounds of the formulas A1 and A2 in which X in each case denotes H are commercially available, for example under the name Precyclemone B.

Compounds of the formula B1 and B2 in which X is H in each case are commercially available, for example under the name Vertomugal, Myrac aldehyde or Citrusal.

Compounds of the formulas C1 and C2 in which X in each case denotes H are commercially available, for example under the name vernaldehyde.

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)

a) binding and / or activating an OR7A5 fragrance receptor or 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 a fusion protein of the invention;

b) to induce or increase the formation of cycloadenosine monophosphate and / or to trigger an influx or increase the concentration of Ca 2+ in a cell containing an OR7A5 fragrance receptor or 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 expressed a fusion protein according to the invention,

c) to increase the flagellum frequency of a sperm,

d) for preparing a medicament for treating a low rate of sperm beating.

To produce an expression system according to the invention, it is expedient to introduce a gene for the receptor through a vector into a host cell introduce. According to another aspect of the invention, therefore, there is provided 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. Such 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.

In particularly preferred embodiments, the vector is an expression vector. Such vectors enable the transcription and translation of the nucleic acid portion encoding the receptor of the invention contained in the vector when the vector is introduced into a suitable host cell. 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. In this context, "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. In this case, 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. Especially Preferred are 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.

Conveniently, the host cell is 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).

According to a further aspect of the invention, 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. 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. In this case, 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. Further advantageous applications of a complex of receptor and agonist or antagonist are described below. Particularly preferred are the complexes of a receptor according to the invention and myracaldehyde (compound of the formula B1 where X = H). These form even at low concentrations of myracaldehyde.

In particular, according to a further aspect of the invention, a biosensor is disclosed, comprising

a) a receptor according to the invention and b) means for detecting a binding of a compound of formula (I) to the receptor, preferably a compound of formula A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 or F2.

The person skilled in the art can detect the binding of the agonist, in particular a compound of the formula (I) and in particular of the formula A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 or F2, to the receptor depending on the type of receptor selected. In a preferred embodiment, 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. By specifically binding the agonist to the receptor, 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. By this conformational change or otherwise, 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. Instead of the cytosolic Ca ²⁺ concentration, it is also possible to measure the increase in concentration or the decrease in concentration of another substance, in particular 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. For this purpose, 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.

Also provided by the invention is a probe for detecting a nucleic acid encoding a portion of a receptor of the invention. Such a probe 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. Instead of such a detectable label, the probe can also be coupled to a solid, for example to metal particles such as magnetic beads (optionally via a linker).

In this connection, 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. Thus, on the one hand, 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. After hybridization, the nucleic acids hybridized with the probe are purified and sequenced.

If only the presence of nucleic acids coding for a fragment of a receptor is to be detected with the probe, 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 To enable nucleic acid, if such a corresponding nucleic acid is present. It is useful if 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. In this case, 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.

Primers for amplifying an OR7A5-encoding nucleic acid are described with the sequences SEQ ID NO. 1 and SEQ ID NO. 2 indicated.

In addition, it is indicated according to the invention to use 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. Thereby, the complexes described above are produced, which have the advantages mentioned above.

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 agonists, ie compounds 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, can therefore also be used to increase the swimming speed of sperm. The addition of an antagonist to sperm leads to the effects described - with constant agonist concentration - either not or not to the usual extent.

Accordingly, the invention also provides a medicament, in particular a contraceptive or contraceptive medicament, comprising as agonist one or more compounds of the formula (I), in particular of the A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 or F2, in particular with X = H, in a pharmaceutically effective amount, in particular in a contraceptive or contraceptive amount. 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. Such a drug utilizes the benefits achievable with the use of agonists and antagonists described above. In addition to the agonists and / or the antagonist, the medicament suitably comprises a pharmaceutically acceptable carrier. Instead of the agonist or antagonist itself, 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. In a contraceptive drug, it may be particularly useful to release 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. It is also possible to provide 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.

In 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.

Furthermore, according to the invention 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. The use of 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.

For producing a fusion protein or expression system according to the invention, a method is disclosed which comprises the steps:

a) introducing a vector according to the invention into a cell, and b) adjusting conditions such that the nucleic acid segment of the vector encoding the receptor is expressed in the cell.

With the aid of this method, it is possible to produce an expression system according to the invention and thus the receptor according to the invention and optionally also a biosensor according to the invention. The receptor according to the invention can be further purified from the cells of the expression system.

According to a further aspect of the invention, a method is provided 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:

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%

Amino acid sequence of the transmembrane domains III to VI of the OR7A5 fragrance receptor or a fusion protein according to the invention,

b) contacting the receptor with the sample to be examined and setting conditions in which a compound of the formula (I) optionally present in the sample binds specifically to the receptor, and

c) Checking whether a compound of the formula (I) has undergone a specific binding with the receptor.

In particular, the receptor may be provided in the form of an expression system in which the receptor is operatively linked to a signal transduction cascade. In this case, 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). However, such an expression system can also be simulated artificially by assembling the receptor and the other components of the signal transduction cascade in vitro.

However, the receptor may also be present as a purified protein bound to the surface of a solid support, particularly a metal surface. In this case, by specifically binding a compound of the formula (I) and / or an antagonist to the receptor, 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.

It is also possible to provide 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.

In particular, 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. The person skilled in the art is familiar with such examination methods under the name "FRET assays" (fluorescence resonance energy transfer assays). Similarly, 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").

In particular, a method for detecting the antagonist in a sample is preferred, 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

Fusion protein,

b) contacting the receptor with the sample to be tested and setting conditions in which an antagonist optionally present in the sample binds specifically to the receptor,

c) contacting the receptor with a compound of formula (I), in particular the A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 or F2, in particular with X = H, to the compound to allow specific binding to the receptor if no antagonist is specifically bound to the receptor, and

d) Check whether the compound of the formula (I) and / or an antagonist has undergone a specific binding with the receptor.

The person skilled in the art recognizes that 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). In particular, the following method embodiments may be appropriate:

In a preferred embodiment, the receptor according to the invention is first provided. For this purpose, 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. Subsequently, the receptor is brought into contact with the optionally antagonist-containing sample. In this case, conditions are set under which an antagonist could specifically bind to the receptor, if it is present in the sample. After this In contact with the antagonist, the receptor is contacted with an agonist. Here, the conditions are adjusted so that the agonist can specifically bind to the receptor if no antagonist has specifically bound to the receptor. Finally, it is checked whether the agonist has bound specifically to the receptor. This can be done, for example, by checking the switching on of a signal transduction cascade with which the receptor is in operative connection. If the agonist has bound to the receptor, then the antagonist is unlikely to be present in a concentration in the sample to be tested that might prevent the specific binding of the agonist. In particular, it may be possible to determine the extent of agonist binding to the receptor and from this - if appropriate after a corresponding calibration - to conclude on the concentration of the antagonist in the sample to be examined.

In a preferred variation of the method, 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.

The invention is explained in more detail below with reference to the figures and the examples, without these being intended to limit the scope of protection of the patent claims.

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. To produce the vector, 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).

The 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 μl of the transfection reagent were added. After 24 h cells were incubated with fresh PBS ⁺⁺ (2.7 mM KCl, 1.5 mM KH ₂ PO ₄ , 137 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 0.9 mM CaCl ₂ , 0.48 mM MgCl ₂ , pH 7.3-7.5) and cultured again with fresh DMEM. For calcium imaging, the DMEM was changed three days after transfection by conventional Ringer's solution. As an indicator of calcium concentration, Fura2 (Molecular Probes) was used according to the manufacturer's instructions.

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.

Example 2: Specificity of sperm fragrance receptors

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.

Figure 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.

In the panels b and c of Figure 2 it is shown that 0R1 D2 transfected cells react with an increase in the intracellular Ca ²⁺ content only when Bourgeonal is administered, but not when myracaldehyde is administered. In addition, the effect of bourgeonal can be reduced by the prior or simultaneous administration of n-undecanal. The skilled person will find further information on the effects of n-undecanal on OR1 D2 in WO 2004 / 033496A1, the content of which is incorporated herein by reference.

The greatest increase in the intracellular Ca 2+ content of OR7A5-transfected HEK293 cells was obtained by administration of myracaldehyde, PI-21788 (compound of formula A1 with X = H) and vernaldehyde.

Example 3: Influence on the Behavior of Sperm

The response of sperm to myracaldehyde was investigated as described in Example 17 of WO 2004 / 033496A1 for Bourgeonal, but fresh semen samples were used instead of frozen semen samples.

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. For each test or control treatment, five repeated experiments were performed using sperm from the five donors. 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 α levels for multiple comparisons.

To study sperm activation, bioassays were performed for the swimming velocity at 37 ^° C. Sperm became one gently Control or test solution of uniform concentration added. The images of sperm (3.0 x 10 ³ cells / ml) were then taken at 90X magnification using a video camera (NEC Model T1 23A) mounted on an Olympus IX70 light microscope. To minimize potential frictional artifacts, the camera had a 100 μm field depth and focused on a region approximately 2 mm from the nearest surface. Sperm video images were digitized at 30 frames / second and processed over 10-second intervals using a CAVMA system (Motion Analysis Corporation, Model VP 320, Expert Vision and Used Software) connected to a Sun SPARC 2 computer workstation (YES Riffell, PJ Krug, RK Zimmer, Journal of Experimental Biology 205, 1439-1450 (2002)). Swimming speed was determined on a frame-by-frame basis and then averaged over each individual path traveled. To avoid measuring horizontally rather than vertically moving cells, short paths with fewer than 11 images were discarded, in which the sperm changed by more than 20% in their apparent size.

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 μL 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. After four hours of incubation at 37 ⁰ C, the content of each capillary was transferred to the well of a toxoplasmosis slide, heat-fixed and stained with 0.1 acridine orange for 1 minute. Cell counts were then made using an Olympus BH2 microscope equipped for phase contrast and epifluorescence applications (JA Riffell, PJ Krug, RK Zimmer, Journal of Experimental Biology 205, 1439-1450 (2002), CC Gee and RK Zimmer-Faust, Journal of Experimental Biology 200, 3185-3192 (1997)), performed at 67x magnification.

10 ^"6 , 10 ^{" 7} , 10 ^"8 or 10 ^{" 9} M myracaldehyde was tested to determine sperm chemoattraction. During the performance of the Chemotaxis bioassays, sperm behavior was recorded on video within a range of 300 μm in front of each capillary tip for 30 seconds. The video began 10 to 15 minutes after the start of the experiment to allow the formation of a chemical gradient. Sperm orientation towards the gradient was quantified using CAVMA. Using circular statistics, the mean vector length (r) and the swim direction were calculated to describe the movement pattern of the sperm examined. The angle of sperm orientation was given in relation to an origin defined as the shortest connection between each individual cell and the capillary tip (0 °). To determine if the cell movement within the chemical gradient was non-random, each mean orientation was compared to a uniform circular distribution using a Ray Ray unimodal assay (JH Zar, Biostatistical Analysis (1984)).

The spermatozoa responded to myracaldehyde (see Figure 3) in a dose-dependent manner by increasing the speed of swimming

Chemotaxis in capillary assays showed. 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

10 nM increased myracaldehyde the swimming velocity of sperm. Furthermore, myracaldehyde showed a chemotactic effect on sperm.

Claims

claims 1. Use of a substance of the formula (I)

(I) in which 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 dashed line drawn in the formula (I) means either a single or a double bond, 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) binding and / or activating an OR7A5 fragrance receptor or 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; b) to induce or increase the formation of cycloadosine monophosphate and / or to trigger an influx or increase the concentration of Ca 2+ in a cell containing an OR7A5 fragrance receptor or 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. c) to increase the flagellum frequency of a sperm, d) for preparing a medicament for treating a low rate of sperm beating. 2. compound selected from the group consisting of

A2 where X = OC ₂ H ₅ ,

B2 where X = OC ₂ H ₅ ,

C1 C2 wherein X = CH ₃ , OCH ₃ or OC ₂ H ₅ ;

D1 D2 wherein, when the compound has the formula D1, X is CH ₃ or OC ₂ H ₅ , and when the compound has the formula D ₂ , X is CH ₃ , OCH ₃ or OC ₂ H ₅ ;

E1 E2 wherein X is OCH ₃ or OC ₂ H ₅ ; and

F1 F2 wherein X is H, CH ₃ , OCH ₃ or OC ₂ H ₅ ; 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 a) the fragrance receptor is heterologous with respect to the cell or b) the fragrance receptor is overexpressed in the cell. The expression system of claim 3, wherein the cell is an optionally degenerate mammalian cell. 5. A vector comprising a nucleic acid portion coding for an OR7A5 fragrance receptor or 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. 6. Nucleic acid consisting of or comprising the sequence SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 4. 7. Use of a nucleic acid according to claim 6 or a nucleic acid of sequence SEQ ID NO 3 for detecting a nucleic acid portion coding for an OR7A5 fragrance receptor or 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 , 8. A biosensor for detecting a compound of the formula (I) according to claim 1, comprising a) an expression system according to any one of claims 3 or 4, and b) means for detecting binding of the compound of formula (I) to said perfume receptor of the expression system. 9. A pharmaceutical composition comprising one or more compounds of the formula (I) according to claim 1 or a salt of the respective compound, in a pharmaceutically effective amount. 10. Use of an OR7A5 fragrance receptor or fragrance receptor having an amino acid sequence homology of at least 70% Amino acid sequence of transmembrane domains III to VI of the OR7A5 fragrance receptor for detecting and / or binding to a compound of formula (I) according to claim 1. 11. A method for detecting a compound of formula (I) according to claim 1, comprising the steps: a) providing an expression system according to one of claims 3 to 4, b) contacting the expression system with a sample optionally containing a said compound of formula (I), and c) checking that the compound of formula (I) has bound to said perfume receptor of the expression system. 12. A method for detecting an antagonist of 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, comprising the steps of: a) providing the receptor according to the invention, b) contacting the receptor with the sample to be tested and setting conditions in which an antagonist optionally present in the sample binds specifically to the receptor, c) contacting the receptor with a compound of formula (I) to give the compound specific binding to the receptor if no antagonist is specifically bound to the receptor, and Check whether the compound of formula (I) and / or an antagonist has specifically bound to the receptor.