EP1516190A1 - Method for identifying agonists or antagonists for the g-protein coupled mas-like 1 receptor - Google Patents

Abstract

Identifying a compound (I) that binds specifically to the G protein-coupled receptor mas-like-1 (R) by (i) treating at least one cell that expresses R on its surface (or a preparation of the cells that contains R) with at least one test compound and (ii) detecting any binding to R. Independent claims are also included for the following: (1) similar methods for identifying compounds that bind specifically to R and are competitive with respect to a first compound, or that bind specifically to R and either activate or inhibit it; (2) pharmaceutical composition containing compounds identified by the new methods; (3) use of substance P (or its cleavage products) or neuropeptide FF (a) for treating diseases associated with defects in R or (b) for preparing a complex with R; and (4) protein that comprises R to which substance P (or its cleavage products) or neuropeptide FF is bound specifically.

Description

description

Method for identifying agonists or antagonists for the G protein-coupled receptor mas like 1

The present invention relates to a method for identifying a compound which changes the activity of the G protein-coupled receptor mas like 1.

G protein-coupled receptors transmit extracellular signals such as from hormones, neurotransmitters, light or odorants via G proteins into the cell interior, whereby different effects can be triggered by an intracellular signal cascade. G proteins usually consist of three different subunits (alpha, beta, gamma). Various heterodimeric G proteins are known, which differ in receptor specificity and effect. The G proteins are activated by GTP. A well-known G protein is the transducin from the vision process.

G protein coupled receptors (GPCR) play an important role in a variety of physiological processes. They represent one of the largest known protein families. It is currently estimated that around 1000 genes of the human genome code for this class of receptors. GPCR are membrane proteins with 7 transmembrane σ helices. A large number of medicinal products work via GPCRs.

GPCRs are particularly involved in signal processing and control of the organism and therefore play a major role in maintaining the function of the intact organism.

The binding of an extracellular ligand leads to a change in the conformation of the GPCR in question. The change in conformation creates the prerequisite for interaction with the associated G protein. The G protein in turn triggers an intracellular signal cascade that is characteristic of the corresponding cell type. The so-called "second messengers" are characteristic of the intracellular signal cascades. These include low-molecular compounds such as, for example, cAMP (cyclic adenosine monophosphate), cGMP (cyclic guanozine monophosphate) or Ca ²⁺ . The intracellular signaling is controlled by changes in the concentration of the second messengers. For this purpose, the G proteins or their subunits interact with proteins such as adenylate cyclase, phospholipase C or ion channels. The change in the concentration of the "second messenger" in turn brings about activation or deactivation of other proteins, in particular kinases and phosphatases. The signal finally ends in a response that is typical for the respective cell assembly, for example the expression of a protein.

The heterotrimeric G proteins are located on the inside of the pias membrane. An activated receptor contacts the G protein heterotimer, which then dissociates a σ subunit and the? Γ complex. Both the activated α subunit and the βγ complex can influence intracellular effector proteins. The G-protein-α subunit family can be divided into different classes. For example, the gas, Gai, Gaq and Ga12 classes are known. GPCRs are classified according to the activated G proteins.

Gs-class GPCRs mediate the stimulation of adenylacyclase and increase the intracellular concentration of cAMP by activating gas. GPCRs of the Gi class inhibit adenylate cyclase by activating Gαi and lower the intracellular cAMP. Gq-class GPCRs, in turn, stimulate various PLCß isoforms by activating Gαq and lead to diacylglycerol and insositol triphosphate (IP3) via the hydrolysis of membrane-bound phosphatidyl-inositol-4,5-biphosphate. IP3 releases Ca ²⁺ from intracellular stores. Most GPCRs can only contact one G-protein-ß-subunit family, ie they have selectivity for a certain signal transduction path. G proteins with changed receptor specificity and different connections to a signal transduction path can be constructed by joining together components from different G proteins to hybrid G proteins using the methods of molecular biology and biochemistry. Hybrid G proteins are fusion constructs that combine sequences of different Gα subunits within one protein. So z. B. by the fusion of the receptor recognition region of Gαi with the effector activation region of Gαq, a Gαq / i hybrid can be produced which receives signals from Gi-coupled receptors but which activates the Gαq-PLCß signal transduction pathway. Such a hybrid, in which the C-terminal 5-amino acids of Gαq has been replaced by the corresponding Gαi sequence (Gαiq5) has been described by Conklin et al. Nature 363, 274-276 (1993) for the first time.

This "coupling" of receptors has the advantage that the end point of the assay (increase in the intracellular Ca ²⁺ concentration compared to the inhibition of the adenylate cyclase) is more easily accessible by measuring techniques and can be used in high throughput screening.

Substance P is a neurotransmitter that is released by both central and peripheral nerve endings of primary afferent neurons. The biological effects of substance P are mediated by tachykinin receptors (= neurokinin receptors), which belong to the group of G protein-coupled receptors. So far, three tachykinin receptors are known: NK1, NK2 and NK3. NK1 preferentially binds substance P, NK2 preferentially binds neurokinin A and NK3 preferentially neurokinin B. However, the endogenous tachykinins are not particularly selective for the respective receptor subtypes and in principle substance P, NKA and NKB activate all three NK receptor subtypes. In addition to the known neurokinin receptor subtypes, another subtype, the NK4 receptor, is proposed.

Substance P is associated with a variety of diseases in which chronic inflammation and pain play a role, such as asthma, chronic bronchitis, inflammatory gastrointestinal diseases, cystitis, but also migraines, depression, vomiting and epileptic diseases. Not least in the cardiovascular area, substance P plays an important role. Substance P is a very powerful vasodilator. Vasodilation and lowering of blood pressure come about through the release of NO from the endothelium of large vessels (mediated via NK1 receptors).

Substance P - acting via neurokinin receptors - is also an important mediator of neurogenic plasma extravasation: Activation of NK1 receptors in endothelial cells of postcapillary venules makes the tight junctions (cell-cell contact) permeable and thus allows the plasma proteins to exit the vascular lumen into the interstitial space. While the role of tachykinins (substance P, neurokinin A and neurokinin B) has been studied fairly well, little is known about the physiological and pathophysiological effects of substance P breakdown products. Substance P is metabolized to N-terminal fragments (1-9,1-7,1-4) and to C-terminal fragments (2-11, 3-11). While the effects of substance P cleavage products on the known neurokinin receptors partly. It has not been previously known whether these cleavage products themselves also bind to receptors and can trigger a signal effect, that is to say act as natural ligands. A ligand is to be understood as a molecule which binds reversibly to a G protein-coupled receptor and exerts an effect on the receptor (stabilization, inactivation, stimulation) via this binding. This effect generally affects a downstream intracellular signal cascade and can be demonstrated using the effects from the signal cascade. A ligand is natural when it is made by a biological system.

The nucleotide and amino acid sequence of the G protein-coupled receptor mas like 1 is known and is available to the public (Genbank: AC 027026).

The nucleotide sequence of mas like 1 is shown in SEQ ID No. 1 and the amino acid sequence of mas like 1 is shown in SEQ ID No. 2.

No natural ligand for the mas like 1 receptor was previously known. This had the disadvantage that no agonists or antagonists for this receptor could be identified. Agonists and antagonists are very useful tools to help a receptor function in terms of its normal activity or pathological studying changed activity. Agonists and antagonists are also advantageous in the restoration of pathologically altered receptor activities, which can manifest themselves, inter alia, in abnormally reduced or increased activity. The object of the present invention is accordingly to provide a method for identifying a compound which acts agonistically or antagonistically on the mas like 1 receptor.

The invention relates to a method for identifying a chemical compound that specifically binds to the G protein-coupled receptor mas like 1, wherein

a] at least one cell is provided which expresses the receptor mas like 1 on its surface, b] at least one chemical compound is provided, c] the cells from a] are brought into contact with the chemical compound from b] under conditions which enable binding to the receptor, d] the binding of the chemical compound to the mas like 1 receptor is determined.

The temperature or the pH value is of particular importance for the production of conditions which enable a chemical compound to bind to the receptor. The temperature is preferably a range from room temperature to 40 ° C., particularly preferably a range from 36 ° C. to 38 ° C. and very particularly preferably 37 ° C. The pH is preferably in the range from 6 to 8 and particularly preferably at pH 7.0.

The invention further relates to a method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1, wherein

a] a preparation of cells that have the receptor mas like 1 on their

Express surface, is provided, in which this receptor is contained, b] at least one chemical compound is provided, c] the cells from a] are brought into contact with the chemical compound from b] under conditions which enable binding to the receptor,

d] the binding of the chemical compound to the receptor mas like 1 is determined.

In a preferred embodiment, the preparation from the cells contains the membrane fraction of a cell extract.

The invention also relates to a method for identifying a chemical compound which binds to the receptor mas like 1 coupled to the G protein in a specific and competitive manner with respect to a first chemical compound, wherein

a] at least one cell is provided which expresses said mas like 1 receptor on its surface, b] at least one first chemical compound is provided which binds to the mas like 1 receptor, c] at least one second chemical compound is provided which differs from the first chemical compound, d] the cells from a] are brought into contact with the first chemical compound from b] and the second chemical compound from c] either simultaneously or in succession under conditions which enable binding to the receptor, e] the binding of the first chemical compound to the receptor or the binding of the second chemical compound to the receptor or the binding of the first and second chemical compounds to the receptor is determined.

In a preferred embodiment herein the first chemical compound consists of substance P or a cleavage product of substance P or the neuropeptide FF. To determine the binding of the first chemical compound and / or the binding of the second chemical compound in the method described above and the further methods of this invention, the creation of a calibration curve may be necessary. The invention further relates to a method for identifying a chemical compound which binds to the receptor mas like 1 coupled to the G protein in a specific and competitive manner with respect to a first chemical compound, wherein

a] a preparation from cells which express the receptor mas like 1 on its surface is provided, in which this receptor is contained, b] at least one first chemical compound is provided which binds to the receptor mas like 1, c] at least a second chemical compound is provided which differs from the first chemical compound, d] the cells from a] are brought into contact with the first chemical compound from b] and the second chemical compound from c] either simultaneously or in succession under conditions, which enable binding to the receptor, e] the binding of the first chemical compound to the receptor or the binding of the second chemical compound to the receptor or the binding of the first and second chemical compounds to the receptor is determined. In a preferred embodiment, the preparation from cells contains the membrane fraction of a cell extract. In a further preferred embodiment, the first chemical compound consists of

Substance P or a cleavage product of substance P or the neuropeptide FF.

The invention also relates to a method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1 and activates it, whereby a] at least one cell is provided which expresses the receptor mas like 1 on its surface and in which a "second messenger" signal cascade can be triggered by means of the receptor mas like 1, b] at least one chemical compound is provided,

c] the cells from a] are brought into contact with the chemical compound from b] under conditions which enable binding to and activation of the receptor, d] the change in the concentration of the "second messenger" in the cell in the presence and Absence of the chemical compound is determined, a change in the presence of the chemical compound indicates activation of the receptor.

The invention further relates to a method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1 and activates it, wherein

a] a preparation from cells which express the mas like 1 receptor on their surface is provided, a “second messenger” signal cascade being able to be triggered in this preparation, b] at least one chemical compound is provided, c] the preparation from cells from a] is brought into contact with the chemical compound from b] under conditions which enable binding to the receptor and activation thereof, d] the change in the concentration of the “second messenger” in the preparation from the cell in the presence and absence of the chemical compound is determined, a change in the presence of the chemical compound indicating activation of the mas like 1 receptor.

In a preferred embodiment, the preparation from the cells contains the membrane fraction of a cell extract. The invention also relates to a method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1 and inhibits it, wherein

a] at least one cell is provided which expresses the receptor mas like 1 on its surface and in which a "second messenger" signal cascade can be triggered by means of the receptor mas like 1, b] at least one chemical compound is provided, c] the cells from a] are brought into contact with the chemical compound from b] under conditions which bind to the receptor and

Inactivation of the same enables d] the change in the concentration of the "second messenger" in the cell to be determined in the presence and absence of the chemical compound, an inactivation of the receptor mas like 1 indicating.

Furthermore, the invention relates to a method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1 and inhibits it, wherein

a] a preparation of cells that have the receptor mas like 1 on their

Express surface, is provided, wherein a "second messenger" signal cascade can be triggered in this preparation, b] at least one chemical compound is provided, c] the preparation of the cells from a] is brought into contact with the chemical compound from b] Conditions that allow binding to and activation of the receptor, d] the change in the concentration of the "second messenger" in the preparation is determined from the cell in the presence and absence of the chemical compound, a change in the presence of the chemical compound Deactivation of the receptor mas like 1 indicates. In a preferred embodiment herein, the preparation from the cells contains the membrane fraction of a cell extract.

The invention also relates to a pharmaceutical preparation containing a compound which has been identified by one of the methods described above, and furthermore auxiliaries for the formulation of a pharmaceutical.

In a preferred embodiment, the pharmaceutical preparation contains substance P or a cleavage product of substance P. Furthermore, the invention comprises the use of substance P or a cleavage product of substance P or the neuropeptide FF for the manufacture of a medicament for the treatment of a disease caused by a malfunction of the Receptor mas like 1 is conditional.

The invention further relates to the use of a compound which has been identified by a method according to the invention for the production of a complex of the mas like 1 receptor with this compound. Suitable compounds for the production of such a complex are, for example, substance P, a cleavage product of substance P or neuropeptide FF. The invention also relates to a complex of the receptor mas like 1 and substance P, a cleavage product of substance P or neuropeptide FF. A complex is to be understood here as a composite of one or more proteins of the receptor in at least specific binding with substance P, a cleavage product of substance P or neuropeptides FF and possibly membrane components or detergents for stabilizing the receptor or receptor / ligand complex. The binding is specific if the binding constant is less than or equal to 100 μM.

The natural ligand can already be present in the biological material or the preparation made of biological material. However, such a ligand can also be added from the outside. For this purpose, the ligand should be present in an amount, concentration and degree of purity such that it binds to the receptor and triggers the receptor signal. The natural ligand is added after the biological material or a preparation of the biological material, which each contain the G protein-coupled receptor mas like 1, is available in a suitable manner (preparation / amount).

Such a natural ligand is preferably substance P, a cleavage product of substance P or neuropeptides FF. The natural ligands and in particular also substance P, a cleavage product of substance P or neuropeptides FF can contain a label which can be detected by a suitable detection method for use in the method according to the invention. Such a label is, for example, a radioactive label or a fluorometrically detectable label.

The method according to the invention is preferably used in such a way that the identified compounds act agonistically or antagonistically to the natural ligand.

The invention also relates to a compound that modifies the activity of the G protein coupled receptor mas like 1, the compound being identified by a method as described above.

Such a compound is characterized in that its mass is preferably between 0.1 to 50 kDa, or particularly preferably between 0.1 to 5 kDa or very particularly preferably between 0.1 to 3 kDa.

Such a compound which has been identified by a method according to the invention can be a protein, an amino acid, a polysaccharide, a sugar, a polynucleotide, a nucleotide, a fatty acid-containing compound, a fat, a fatty acid, a fatty acid derivative or an aromatic hydrocarbon compound.

The invention further relates to a medicament which contains a compound which has been identified by a method according to the invention, and furthermore Formulation aids for a drug and / or other additives. This medicine is particularly suitable for the treatment of cardiovascular disease.

A compound that has been identified by a method according to the invention can be used to produce a medicament for the treatment of cardiovascular disease.

The G protein-coupled receptor mas like 1 for carrying out the method according to the invention can be produced by expressing an exogenous DNA sequence in a prokaryote or eukaryote. In principle, any prokaryotic or eukaryotic plasmid vector, bacteriophage vector or yeast plasmid vector is suitable for producing the protein. Examples of such vectors are pBR322, pUC18,19, pBlueScript, pcDNA3.1 and others. Expression can be transient or permanent.

Recombinant vector constructions can be carried out with the help of the relevant specialist knowledge, as shown, for example, in “F.M. Ausubel et al., Current Protocols in Molecular Biology, Wiley & Sons, New York (ISBN 0-471 -50338-X) "or om" J. Sambrook, EF Fritsch, T. Mamiatis, Molecular Cloning, second edition, Cold Spring Harbor Laboratory Press "(ISBN 0-87969-309-6). In this case, a polynucleotide coding for an amino acid sequence according to one of the described sequence information (SEQ ID NO. 1) or a polynucleotide sequence according to one of the described sequence information (SEQ ID NO. 2) is built into a basic vector. A base vector is to be understood as a vector in which a polynucleotide sequence of a polynucleotide by means of the methods of

Molecular biology can be built in and cloned in a microorganism, for example a bacterium, fungus or the cell of a cell culture. The base vector can consist, for example, of a plasmid with an antibiotic resistance marker, an "origin of replication" suitable for multiplication of the plasmid in bacteria or cell cultures, and a promoter suitable for expression of a protein. The base vector can also consist of a phage vector, a phagemid vector, for example Phasmid vector, a cosmid vector, a virus vector, a YAC vector or other type of vector. Examples of basic vectors are pUC 18, pUC19, pBLuescript, pKS, pSK and others. The polynucleotide to be incorporated is incorporated via suitable restriction interfaces using the appropriate restriction enzymes, which are commercially available from companies such as BioLabs, Röche Diagnostics, Stratagene and others. Such restriction sites can be, for example, the recognition sites of the restriction enzymes BamHI, EcoRI, Sall, EcoRV and others.

In a preferred embodiment, the recombinant vector construction consists of an expression vector which can be used in eukaryotes and / or prokaryotes. An expression vector contains a promoter which can be functionally linked to a polynucleotide sequence so that a protein encoded by this polynucleotide sequence is synthesized in a biological organism, for example a bacterium, fungus or the cell of a eukaryotic cell line. The promoter can be inducible, for example by means of

Tryptophan or it can be constitutively active. Examples of expression vectors are pUC18, pUC19, pBluescript, pcDNA3.1 or others.

Biological material is any material that contains genetic information that can reproduce itself or can be reproduced in a biological system. Biological material is, for example, cells from human or animal tissues or organs such as in particular the brain, adipose tissue, lungs, heart, liver, kidney, spleen, muscle and others. Biological material is also, for example, bacteria or fungi such as Escherichia coli or Saccharomyces cerevisiae. Biological material also includes cells from cell cultures.

In the case of cells from animal or human tissues, biological material can be obtained by biopsy, surgical removal, removal by means of syringes or catheters or comparable techniques. The cells removed in this way can be frozen, processed or taken in cell culture. Bacteria and yeast cells are propagated and processed using common microbiology techniques. Appropriate instructions for this can be found in “Current Protocols in Molecular Biology; ed .: FM Ansubel et al., constantly renewed, edition 2001, publisher John Wiley & Sons ".

Biological material can also consist of the cells of an animal cell culture. Such cells are, for example, mouse cells, rat cells or hamster cells. The cell culture cells can be primary cell types or established cell lines. Examples of established cell lines are mouse 3T3 cells, CHO cells, HEK cells or Heia cells. Maintaining, cultivating and multiplying cell lines is described in standard textbooks, such as, for example, in “Basic Cell Culture; ed .: J.M. Davis IRL Press, Oxford (1996).

A preparation of a biological material is produced, for example, by digestion of the biological material and subsequent cleaning steps. Methods for disintegrating the biological material can in particular be repeated freezing and thawing, treatment with ultrasound, the use of a French press, the addition of detergents and enzymes or the like. Subsequent purification steps consist, for example, of differential centrifugation, precipitation with ammonium sulfate or organic solvents, the use of chromatographic techniques and others. Chromatographic techniques are, for example

Polyacrylamide gel electrophoresis, high pressure liquid chromatography, ion exchange chromatography, affinity chromatography, gas chromatography, mass spectrometry and others. For this purpose and in particular also for detailed instructions for the pure presentation of proteins, detailed instructions are available to the person skilled in the art in textbooks, in particular in “Current Protocols in Protein Science, ed .: J.E. Coligan et al., 2001 edition, published by John Wiley & Sons.

The biological material or the preparation made of biological material can be brought into contact with a chemical compound in conventional laboratory vessels such as Eppendorf tubes, centrifuge tubes or glass flasks. The underlying aqueous medium contains, for example, buffer substances, nutrient medium components, monovalent or divalent ions such as Na ⁺ , K \ Ca ²⁺ , CI ^" , SO ₄ ^2" , PO ₃ ^{2 "} or others, furthermore proteins, glycerol or others. For contacting certain constant conditions such as in particular the temperature, the pH value, the ion conditions , the concentration of a protein, the volume or other factors can be advantageous, for example by contacting in incubators at constant temperature, in the presence of a buffer or with the precisely weighed amounts of ions or proteins in particular also contain a certain proportion of an organic solvent such as dimethyl sulfoxide, methanol or ethanol, but the content of such a solvent is preferably not more than 10% by volume of the batch.

A chemical compound is provided, for example, by chemical synthesis. The standard synthetic methods are familiar to the person skilled in the art. The chemical compound can be part of a collection of chemical compounds, such as arise from the storage and cataloging of chemical compounds from completed synthesis programs (so-called "chemical libraries"). In other cases, the compound can be from a microorganism, in particular a bacterium, but also from a fungus or a plant (natural product).

Suitable pharmaceutical compounds can be administered as pharmaceuticals in oral, oral, topical, parenteral or rectal form. The most suitable route of administration depends in each individual case on the type and severity of the condition to be treated and on the type of the compound used in each case. A suitable active ingredient concentration is approximately 1% to 35%, preferably approximately 3% to 15%.

Examples

Cloning of the human mas like 1 1 receptor

The sequence of the human GPCR mas like 1 is stored in databases that are open to the public. The corresponding access number from Genbank is AC 027026. SEQ ID No. 1 contains the nucleotide sequence of the mas like 1 receptor. The corresponding amino acid sequence can be found in SEQ ID No. 2. The human gene contains no introns. The receptor was therefore replicated from genomic DNA using a polymerase chain reaction (PCR). In the present case, the human genomic DNA came from the company Clontech, Palo Alto. In principle, human genomic DNA from another company or from our own production is also suitable for carrying out the PCR. The PCR reaction conditions were chosen as follows: first incubation at 94 ° C for

10 10 min., Then 35 cycles per cycle, incubation at 94 ° C. for 1 min., Then at 60 ° C. for 1 min. And finally at 72 ° C. for 2 min. The reaction was carried out using the GC-Melt Kit from the company Clontech performed. Another protocol used in the textbooks is also suitable for carrying out the PCR, in particular with regard to buffer conditions and enzyme. The primers used according to SEQ ID No. 3 and

15 4 were constructed so that a Hindlll site (SEQ ID No. 3) or an EcoRI interface (SEQ ID No. 4) was available. The PCR product formed was 1260 base pairs in length. It was inserted into the expression vector pcDNA3.1 using the Hindi II and EcoRI interfaces. This vector is commercially available, inter alia, from Invitrogen, Carlsbad in California, USA. 0

Quantitative RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) in real time using Taqman

Commercially available RNA samples from the following tissues and organs were available: 5 adrenal gland, fetal brain, fetal liver, small intestine, heart, kidney, liver, lung, pancreas, placenta, prostate, skeletal muscle, spleen, testicles, thymus, bone marrow, salivary gland, Thymus, trachea and uterus.

The RNA samples were first subjected to DNAase 0 digestion under standard reaction conditions. Approximately 2 μg of the RNA treated with RNA was used to produce cDNA using a reverse transcriptase from MMLV Moloney Murine Leukemia Virus. The reaction mixture for this was adjusted to a pH of 7.0 and contained 25 mM / MgCI ₂ each 10 mM dNTPs, 50 μM random hexamer primer, 50 μM oligo (dT) ι ₆ and 20 U / μl RNase inhibitor. The reaction was carried out in a volume of 100 μl, incubating first for 10 minutes at 25 ° C., then for 60 minutes at 42 ° C., then inactivating for 5 minutes at 95 ° C. and finally cooling in ice 5 has been.

The subsequent PCR reaction was carried out using a TaqMan Universal PCR Master Mix from Applera from Weiterstadt, Germany. With regard to buffers, enzyme equipment and other reagents, this corresponds to the usual laboratory protocols known to those skilled in the art.

The following sequences were used as primers: 5'-TGC TTT GTG GCA AGG AGA CC-3 '(SEQ ID No. 5; "forward" primer) 15

5'-TTC CTA CCA GCC CGA CCA G-3 '(SEQ ID No. 6; "reverse" primer).

The incubation conditions for the PCR were chosen as follows: 2 min. 20 50 ° C., then 10 min. 95 ° C., then 40 cycles for each cycle, 15 seconds 95 ° C. and 1 min. 60 ° C. The primers were used in a concentration of 50 μM each. Ribosomal 18 S RNA was used as a control.

The quantitative analysis was carried out using a fluorescence resonance energy transfer 25 (FRET). The person skilled in the art knows the quantitative determination of this type also under the name Taqman-PCR. A Taqman sample consists of a single-stranded oligonucleotide, which is labeled with two different fluorophores at the ends. The fluorophore of the 3 'end, the so-called acceptor, functions with a weakening effect ("quencher") of the 30 fluorophore from the 5' end, the so-called donor.

The 5 'end donor is released by the 5' exonuclease activity of Taq DNA polymerase in the course of the chain extension reaction. Since the donor in free form is no longer quenched, its amount can be determined using a fluorimeter. Since the amount of donor formed and the accumulated PCR product behave proportionally, the amplified DNA can be quantified. It is important to ensure that the melting temperature of the Taqman sample is higher than that of the primers for the amplification.

FAM (6-carboxyfluorescein) was used as the fluorophore as the donor and TAMRA (5-carboxy-tetramethylrhodamine) as the acceptor (= quencher). The Taqman sample used had the following nucleotide sequence:

3'-TGA TCC CGG TCT TCC TGA TCC TTT TCA-5 '(SEQ ID No. 7).

Cell culture and transfection for the FLIPR assay

CHO-K1 cells were cultured in basal Iscove medium, which was supplemented with 10% fetal bovine serum 10,000 U / ml - 10,000 μg / ml penicillin-streptomycin, gentamycin and 2 mM glutamine.

Iscove-Medium is commercially available from, among others, the company Biochrom, Berlin. The composition of Iscove medium is described in "Iscove, NN, Melchers, F., Journal of Experimental Medicine 147, 923-933 (1978)". The CHO-K1 cells were used in an amount of 2 × 10 for the transient transfection ⁵ cells were placed on a 35 mm culture dish after approximately 24 hours of incubation or at a confluence of 50-80% with 2 μg DNA using Lipofectamine reagent, which is commercially available from Invitrogen Life Technologies, Karlsruhe, among others. transfected.

About 16 to 18 hours after the transfection, the cells were transferred to 96 well microtiter plates with a density of about 80,000 cells per well. The mixture was then incubated for a further 18 to 24 hours. 95 μl of HBSSC (Hank's buffered Saline Solution), which contained 20 mM HEPES, 2.5 mM sample oath, 4 μM Fluo4 and 1% fetal bovine serum, were then added and incubated for 1 hour at 37 ° C. in the presence of 5% CO ₂ . Fluo4 is a dye that fluoresces in the presence of Ca ²⁺ . Fluo4 is commercially available from Molecular Probes Europe BV, Leiden, among others distributed. After the incubation, the cells were washed three times with PBS (phosphate buffered saline), which contained 1 mM MgCl ₂ , 1 mM EDTA and 2.5 mM sample oath.

5 After the last washing step, about 100 μl of the solution was left on the cells in each well. The peptides to be tested were dissolved in water or DMSO (dimethyl sulfoxide) in a concentration of 2 mM. The corresponding dilutions of the final concentration (20 mM) were prepared from such a stock solution. The fluorescence was measured using a FLIPR

10 (Fluorometric Imaging Plate Reader; Molecular Devices GmbH, Ismaning) measured for a total of 3 minutes in 1 second intervals during the first minute and in 3 second intervals during the second and third minutes. The measured values between 18 seconds and 37 seconds were used to determine the agonistic effect.

15

Identification of ligands of the mas like 1 receptor

Various peptides were tested for their stimulating properties on the G protein-coupled receptor mas like 1. The test was carried out using FLIPR

20 technology implemented. For this purpose, the receptor was transiently expressed in CHO-K1 cells.

The following peptides were tested: neuropeptide FF, neuropeptide AF, neuropeptide SF, angiotensin, bradykinin, bombesin, adrenomedullin, substance P, substance P1-9, substance P2-11.

25 Fluorescence formation in the cells was detected in the presence of substance P (approx. 500 fluorescence intensity units), substance P2-11 (approx. 2500 fluorescence intensity units) and neuropeptide FF (approx. 2000 fluorescence intensity units). The zero value was approx. 20 fluorescence intensity units. The activation of the receptor showed in the concentration range of 5 x 10 ^"6 to 1 x

30 10 ^"5 M of substance P and substance P2-11 a linear dependency. The EC ₅ for substance P was 10.37 μM. The substance P2-11 released arachidonic acid in the transiently transfected CHO-K1 cells.

In this way, the substance P, the substance P2-11 and the neuropeptide FF could be identified as natural ligands for the G protein-coupled receptor called mas like 1.

A test system constructed according to the detection method for finding agonists or antagonists with regard to the activity of mas like 1 additionally contained substance P, substance P2-11 or neuropeptide FF. Other fragments of substance P, such as substance P4-11 or substance P1-9, had an agonistic effect.

Neuropeptide FF has the following amino acid sequence:

H-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Pro-NH ₂ . (SEQ ID No. 8)

Substance P2-11 has the following amino acid sequence:

H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ₂ . (SEQ ID No. 9).

Claims

claims: 1. A method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1, wherein a] at least one cell is provided which expresses the receptor mas like 1 on its surface, b] at least one chemical compound is provided, c] the cells from a] are brought into contact with the chemical compound from b] under conditions which enable binding to the receptor, d] the binding of the chemical compound to the mas like 1 receptor is determined. 2. A method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1, where a] a preparation of cells which express the receptor mas like 1 on their surface is provided, in which this receptor is contained, b] at least one chemical compound is provided, c] the cells from a] with the chemical compound from b ] are brought into contact under conditions which enable binding to the receptor, d] the binding of the chemical compound to the mas like 1 receptor is determined. 3. The method according to claim 2, characterized in that the preparation from cells contains the membrane fraction of a cell extract. 4. A method for identifying a chemical compound which binds to the receptor mas like 1 coupled to the G protein in a specific manner and competitively with respect to a first chemical compound, wherein a] at least one cell is provided which expresses said mas like 1 receptor on its surface, b] at least one first chemical compound is provided which binds to the mas like 1 receptor, c] at least one second chemical compound is provided which differs from the first chemical compound, d] the cells from a] are brought into contact with the first chemical compound from b] and the second chemical compound from c] either simultaneously or in succession under conditions which enable binding to the receptor, e] the binding of the first chemical compound to the receptor or the binding of the second chemical compound to the receptor or the binding of the first and second chemical compounds to the receptor is determined. 5. The method according to claim 4, characterized in that the first chemical compound is the substance P or a cleavage product of the substance P or the neuropeptide FF. 6. A method for identifying a chemical compound which binds to the receptor mas like 1 coupled to the G protein in a specific and competitive manner with respect to a first chemical compound, wherein a] a preparation is provided from cells which express the receptor mas like 1 on their surface, in which this receptor is contained, b] at least one first chemical compound is provided which binds to the receptor mas like 1, c] at least one second chemical compound is provided which differs from the first chemical compound, d] the cells from a] are brought into contact with the first chemical compound from b] and the second chemical compound from c] either simultaneously or in succession under conditions that a Enable binding to the receptor, e] the binding of the first chemical compound to the receptor or the binding of the second chemical compound to the receptor or the binding of the first and second chemical compounds to the receptor is determined. 7. The method according to claim 6, characterized in that the preparation from cells contains the membrane fraction of a cell extract. 8. The method according to claim 7, characterized in that the first chemical compound is the substance P or a cleavage product of the substance P or the neuropeptide FF. 9. A method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1 and activates it, wherein a] at least one cell is provided which expresses the receptor mas like 1 on its surface and in which a "second messenger" signal cascade can be triggered by means of the receptor mas like 1, b] at least one chemical compound is provided, c] the cells from a] are brought into contact with the chemical compound from b] under conditions which enable binding to the receptor and activation thereof, d] the change in the concentration of the “second messenger” in the cell in Presence and absence of the chemical compound determined a change in the presence of the chemical compound indicates activation of the mas like 1 receptor. 10. A method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1 and activates it, wherein a] a preparation from cells which express the mas like 1 receptor on their surface is provided, a “second messenger” signal cascade being able to be triggered in this preparation, b] at least one chemical compound is provided, c] the preparation from cells from a] is brought into contact with the chemical compound from b] under conditions which enable binding to the receptor and activation thereof, d] the change in the concentration of the "second messenger" in the Preparation from the cell is determined in the presence or absence of the chemical compound, a change in the presence of the chemical compound indicating activation of the mas like 1 receptor. 11. The method according to claim 10, characterized in that the preparation from cells contains the membrane fraction of a cell extract. 12. A method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1 and inhibits it, wherein a] at least one cell is provided which expresses the receptor mas like 1 on its surface and in which a "second messenger" signal cascade can be triggered by means of the receptor mas like 1, b] at least one chemical compound is provided, c] the cells from a] are brought into contact with the chemical compound from b] under conditions which enable binding to the receptor and inactivation thereof, d] the change in the concentration of the "second messenger" in the cell in the presence or Absence of the chemical compound is determined, a change in the presence of the chemical compound indicates an inactivation of the mas like 1 receptor. 13. A method for identifying a chemical compound which specifically binds to the G protein-coupled receptor mas like 1 and inhibits it, wherein a] a preparation of cells which express the mas like 1 receptor on their surface is provided, a “second messenger” signal cascade being triggered in this preparation, b] at least one chemical compound is provided, c] the preparation of the cells from a] is brought into contact with the chemical compound from b] under conditions which enable binding to the receptor and activation thereof, d] the change in the concentration of the "second messenger" in the Preparation from the cell is determined in the presence or absence of the chemical compound, a change in the presence of the chemical compound indicating a deactivation of the mas like 1 receptor. 14. The method according to claim 13, characterized in that the preparation from cells contains the membrane fraction of a cell extract. 15. The method according to claim 1, 4, 5, 9, 10 characterized in that the cell is a CHO, CHO-K1 or a HEK cell. 16. Pharmaceutical preparation containing a compound, which by a Method according to one or more of claims 1 to 14 has been identified, and auxiliaries for the formulation of a pharmaceutical. 5 17. Medicament preparation according to claim 15 containing substance P or a cleavage product of substance P or neuropeptides FF. 18. Use of substance P for the manufacture of a medicament for the treatment of a disease caused by a malfunction of the receptor mas 10 like 1 is conditional. 19. Use of a fission product of substance P for the manufacture of a medicament for the treatment of a disease which is caused by malfunction of the mas like 1 receptor. 15 20. Use of Neuropeptide FF for the manufacture of a medicament for the treatment of a disease which is caused by malfunction of the mas like 1 receptor. 20 21. Use of substance P or a cleavage product of substance P or the neuropeptide FF for the production of a complex of the mas like 1 receptor with this compound. 22. Protein containing the mas like 1 receptor, characterized in that the substance P or a cleavage product of the substance P or the neuropeptide FF is specifically bound to the mas like 1 receptor.