CA2488883A1 - Method for identifying agonists or antagonists for the gpr45-like/gpr63 receptor - Google Patents
Method for identifying agonists or antagonists for the gpr45-like/gpr63 receptor Download PDFInfo
- Publication number
- CA2488883A1 CA2488883A1 CA002488883A CA2488883A CA2488883A1 CA 2488883 A1 CA2488883 A1 CA 2488883A1 CA 002488883 A CA002488883 A CA 002488883A CA 2488883 A CA2488883 A CA 2488883A CA 2488883 A1 CA2488883 A1 CA 2488883A1
- Authority
- CA
- Canada
- Prior art keywords
- receptor
- compound
- protein
- gpr63
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4719—G-proteins
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Heart & Thoracic Surgery (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cardiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for identifying a compound, which modifies the activity of the G-protein coupled GPR45-like/GPR63 receptor.
Description
WO 03/104.480 PCTIEP03105600 Method for identifying agonists or antagonists of the GPR45 IikeIGPR63 receptor Description The present invention relates to a method for identifying a compound which modifies the activity of the G protein-coupled receptor GPR45 IikeIGPR63.
G protein-coupled receptors mediate extracellular signals such as, for example, from hormones, neurotransmitters, light or odorants via G proteins into the interior of the cell, and various effects can be initiated via an intracellular signal cascade. G
proteins normally consist of three different subunits (alpha, beta, gamma).
Various heterodimeric G proteins which differ in receptor specificity and effect are known.
The G proteins are activated by GTP. A well-known G protein is transducin from the vision process.
G protein-coupled receptors (GPCR) play an important part in a large number of physiological processes. They are one of the largest protein families known.
It is currently estimated that about 1 000 genes in the human genome code for this class of receptors. GPCR are membrane proteins with 7 transmembrane a-helices. A
large number of medicaments displays its effect via GPCRs.
GPCRs are involved especially in signal processing and control of the organism and therefore play a superordinate part in maintaining the function of the intact organism.
The binding of an extracellular ligand leads to a conformational change in the relevant GPCR. The conformational change creates the preconditions for interaction with the respectively associated G protein. The G protein in turn initiates an intracellular signal cascade which is characteristic of the relevant cell type.
The so-called second messengers are characteristic of intracellular signal cascades.
By these are meant low molecular weight compounds such as, for example, cAMP
(cyclic adenosine monophosphate), cGMP (cyclic guanozine monophosphate) or Ca2+. The intracellular signaling is controlled by changes in the concentration of the second messengers. The G proteins and their subunits interact for this purpose with proteins such as adenylate cyclase, phospholipase C or ion channels. The change in the concentration of the second messenger in turn brings about an activation or inactivation of other proteins, especially of kinases and phosphatases. The signal finally terminates in a response typical of the particular cell assembly, for example the expression of a protein.
The heterotrimeric G proteins are located on the inside of the plasmembrane.
An activated receptor makes contact with the G protein heterotimer, which then dissociates an a subunit and the ~iy complex. Both the activated a subunit and the ~iy complex are able to influence intracellular effector proteins. The G protein a subunit fariiily can be divided into various classes. Known examples are the Gas, Gai, Gaq and Ga12 classes. GPCRs are classified according to the activated G proteins.
GPCRs of the Gs class mediate, via activation of Gas, the stimulation of adenyla-cyclase and increase the intracellular cAMP concentration. GPCRs of the Gi class bring about, via activation of Gal, an inhibition of adenylate cyclase and reduce the intracellular cAMP.
GPCRs of the Gq class in turn achieve, via activation of Gaq, a stimulation of various PLC isoforms and lead, via hydrolysis of membrane-bound phosphatidylinositol 4,5-biphosphate, to diacylglycerol and inositol triphosphate (IP3). IP3 releases Ca2+
from intracellular stores. Most GPCRs are able to make contact with only one G
protein ~i subunit family, i.e. they have selectivity for a particular signal transduction pathway.
G proteins with altered receptor specificity and different attachment to a signal transduction pathway can be constructed by joining together components from different G proteins to give hybrid G proteins by the methods of molecular biology and biochemistry.
Hybrid G proteins are fusion constructs which combine within one protein sequences of different Ga subunits. Thus, for example, it is possible by fusing the receptor recognition region of Gai with the effector activation region of Gaq to produce a Gaq/i hybrid which receives the signals of Gi-coupled receptors but switches on the Gaq PLC~3 signal transduction pathway. Such a hybrid in which the C-terminal 5-amino acids of Gaq has been replaced by the corresponding Gai sequence (GaiqS) was described for the first time by Conklin et al. Nature 363, 274-276 (1993).
This "rerouting" of receptors has the advantage that the assay endpoint (increase in intracellular Ca2+ concentration compared with inhibition of adenylate cyclase) is more easily accessible by measurement techniques and can be used in high troughput screening.
Sphingosine 1-phosphate (S1 P) and lysophosphatidic acid (LPA)) are lipid signal molecules which are formed from membrane phospholipids. S1 P and LPA are mainly known as intracellular signal molecules. In the case of some GPCRs, S1 P or LPA functions as natural ligand. A ligand means a molecule which reversibly binds to a G protein-coupled receptor and exerts via this binding an effect on the receptor (stabilization, inactivation, stimulation). This effect generally relates to a downstream intracellular signal cascade and can be detected from the effects on the signal cascade. A ligand is natural if it is produced by a biological system.
The nucleotide sequence and amino acid sequence of the GPR451ikeIGPR63 receptor is known (Genbank: NM 030784; TREMBL:Q9b2i6). The nucleotide sequence of the GPR451ike/GPR63 receptor is depicted in SEQ ID NO. 1, and its amino acid sequence is depicted in SEQ ID NO. 2. No natural ligand for the GPR451ikeIGPR63 receptor was previously known. It is thus not possible to identify any agonists or antagonists for this receptor function. Agonists or antagonists are usually defined organic molecules with a precise structure and a reproducible process of preparation. Only with the aid of such compounds is it possible to investigate the function of this receptor in various tissues, stages of development and different external influences. Aids allowing the roll of this receptor in normal and pathologically altered tissues are thereby provided. It is therefore an object of the present invention accordingly to provide a method for identifying a compound which has an agonistic or antagonistic effect on the GPR451ike/GPR63.
The invention relates to a method for identifying a compound which modifies the activity of the G protein-coupled receptor GPR451ikeIGPR63, where a] biological material or a preparation of biological material which contains the G
protein-coupled receptor GPR451ike/GPR63 and a natural ligand of this receptor is provided, b] a chemical compound is provided;
c] the biological material or the preparation of biological material from a]
is brought into contact with the chemical compound from b]
d] the activity of the G protein-coupled receptor GPR451ikeIGPR63 is determined.
The modification of the activity of the G protein-coupled receptor GPR451ike/GPR63 consists, in particular, of its stabilization, switching on, switching off, elevation or depression.
The bringing into contact of the chemical compound with the biological material or the preparation thereof takes place under conditions which make an interaction possible. Important in this connection is, in particular, the temperature (room temperature to about 37°C) and the pH (pH 6-8, preferably pH about 7.0).
In a preferred embodiment of the method, a G protein-coupled receptor GPR451ike/GPR63 is selected from one of the following groups a] a receptor consisting of the amino acid sequence as shown in SEQ ID NO. 2, b] a receptor which is lacking one or more amino acids in relation to the amino acid sequence of SEQ ID NO. 2, c] a receptor in which one or more amino acids have been added in relation to the amino acid sequence of SEQ ID NO. 2, d] a receptor in which one or more amino acids have been replaced by other amino acids in relation to the amino acid sequence of SEQ ID NO. 2.
The G protein-coupled receptor GPR451ike/GPR63 for carrying out the method described above can preferably be encoded from one of the following groups:
G protein-coupled receptors mediate extracellular signals such as, for example, from hormones, neurotransmitters, light or odorants via G proteins into the interior of the cell, and various effects can be initiated via an intracellular signal cascade. G
proteins normally consist of three different subunits (alpha, beta, gamma).
Various heterodimeric G proteins which differ in receptor specificity and effect are known.
The G proteins are activated by GTP. A well-known G protein is transducin from the vision process.
G protein-coupled receptors (GPCR) play an important part in a large number of physiological processes. They are one of the largest protein families known.
It is currently estimated that about 1 000 genes in the human genome code for this class of receptors. GPCR are membrane proteins with 7 transmembrane a-helices. A
large number of medicaments displays its effect via GPCRs.
GPCRs are involved especially in signal processing and control of the organism and therefore play a superordinate part in maintaining the function of the intact organism.
The binding of an extracellular ligand leads to a conformational change in the relevant GPCR. The conformational change creates the preconditions for interaction with the respectively associated G protein. The G protein in turn initiates an intracellular signal cascade which is characteristic of the relevant cell type.
The so-called second messengers are characteristic of intracellular signal cascades.
By these are meant low molecular weight compounds such as, for example, cAMP
(cyclic adenosine monophosphate), cGMP (cyclic guanozine monophosphate) or Ca2+. The intracellular signaling is controlled by changes in the concentration of the second messengers. The G proteins and their subunits interact for this purpose with proteins such as adenylate cyclase, phospholipase C or ion channels. The change in the concentration of the second messenger in turn brings about an activation or inactivation of other proteins, especially of kinases and phosphatases. The signal finally terminates in a response typical of the particular cell assembly, for example the expression of a protein.
The heterotrimeric G proteins are located on the inside of the plasmembrane.
An activated receptor makes contact with the G protein heterotimer, which then dissociates an a subunit and the ~iy complex. Both the activated a subunit and the ~iy complex are able to influence intracellular effector proteins. The G protein a subunit fariiily can be divided into various classes. Known examples are the Gas, Gai, Gaq and Ga12 classes. GPCRs are classified according to the activated G proteins.
GPCRs of the Gs class mediate, via activation of Gas, the stimulation of adenyla-cyclase and increase the intracellular cAMP concentration. GPCRs of the Gi class bring about, via activation of Gal, an inhibition of adenylate cyclase and reduce the intracellular cAMP.
GPCRs of the Gq class in turn achieve, via activation of Gaq, a stimulation of various PLC isoforms and lead, via hydrolysis of membrane-bound phosphatidylinositol 4,5-biphosphate, to diacylglycerol and inositol triphosphate (IP3). IP3 releases Ca2+
from intracellular stores. Most GPCRs are able to make contact with only one G
protein ~i subunit family, i.e. they have selectivity for a particular signal transduction pathway.
G proteins with altered receptor specificity and different attachment to a signal transduction pathway can be constructed by joining together components from different G proteins to give hybrid G proteins by the methods of molecular biology and biochemistry.
Hybrid G proteins are fusion constructs which combine within one protein sequences of different Ga subunits. Thus, for example, it is possible by fusing the receptor recognition region of Gai with the effector activation region of Gaq to produce a Gaq/i hybrid which receives the signals of Gi-coupled receptors but switches on the Gaq PLC~3 signal transduction pathway. Such a hybrid in which the C-terminal 5-amino acids of Gaq has been replaced by the corresponding Gai sequence (GaiqS) was described for the first time by Conklin et al. Nature 363, 274-276 (1993).
This "rerouting" of receptors has the advantage that the assay endpoint (increase in intracellular Ca2+ concentration compared with inhibition of adenylate cyclase) is more easily accessible by measurement techniques and can be used in high troughput screening.
Sphingosine 1-phosphate (S1 P) and lysophosphatidic acid (LPA)) are lipid signal molecules which are formed from membrane phospholipids. S1 P and LPA are mainly known as intracellular signal molecules. In the case of some GPCRs, S1 P or LPA functions as natural ligand. A ligand means a molecule which reversibly binds to a G protein-coupled receptor and exerts via this binding an effect on the receptor (stabilization, inactivation, stimulation). This effect generally relates to a downstream intracellular signal cascade and can be detected from the effects on the signal cascade. A ligand is natural if it is produced by a biological system.
The nucleotide sequence and amino acid sequence of the GPR451ikeIGPR63 receptor is known (Genbank: NM 030784; TREMBL:Q9b2i6). The nucleotide sequence of the GPR451ike/GPR63 receptor is depicted in SEQ ID NO. 1, and its amino acid sequence is depicted in SEQ ID NO. 2. No natural ligand for the GPR451ikeIGPR63 receptor was previously known. It is thus not possible to identify any agonists or antagonists for this receptor function. Agonists or antagonists are usually defined organic molecules with a precise structure and a reproducible process of preparation. Only with the aid of such compounds is it possible to investigate the function of this receptor in various tissues, stages of development and different external influences. Aids allowing the roll of this receptor in normal and pathologically altered tissues are thereby provided. It is therefore an object of the present invention accordingly to provide a method for identifying a compound which has an agonistic or antagonistic effect on the GPR451ike/GPR63.
The invention relates to a method for identifying a compound which modifies the activity of the G protein-coupled receptor GPR451ikeIGPR63, where a] biological material or a preparation of biological material which contains the G
protein-coupled receptor GPR451ike/GPR63 and a natural ligand of this receptor is provided, b] a chemical compound is provided;
c] the biological material or the preparation of biological material from a]
is brought into contact with the chemical compound from b]
d] the activity of the G protein-coupled receptor GPR451ikeIGPR63 is determined.
The modification of the activity of the G protein-coupled receptor GPR451ike/GPR63 consists, in particular, of its stabilization, switching on, switching off, elevation or depression.
The bringing into contact of the chemical compound with the biological material or the preparation thereof takes place under conditions which make an interaction possible. Important in this connection is, in particular, the temperature (room temperature to about 37°C) and the pH (pH 6-8, preferably pH about 7.0).
In a preferred embodiment of the method, a G protein-coupled receptor GPR451ike/GPR63 is selected from one of the following groups a] a receptor consisting of the amino acid sequence as shown in SEQ ID NO. 2, b] a receptor which is lacking one or more amino acids in relation to the amino acid sequence of SEQ ID NO. 2, c] a receptor in which one or more amino acids have been added in relation to the amino acid sequence of SEQ ID NO. 2, d] a receptor in which one or more amino acids have been replaced by other amino acids in relation to the amino acid sequence of SEQ ID NO. 2.
The G protein-coupled receptor GPR451ike/GPR63 for carrying out the method described above can preferably be encoded from one of the following groups:
5 a] a polynucleotide sequence as shown in SEQ ID NO. 1, b] a polynucleotide sequence as shown in SEQ ID NO. 1 in which, owing to the degeneracy of the genetic code, one or more of the nucleotide positions contain a different nucleotide, c] ~ a polynucleotide sequence which hybridizes under stringent conditions with a polynucleotide sequence consisting of SEQ ID NO. 1.
~ The G protein-coupled receptor GPR451ikeIGPR63 for carrying out the method of the invention can be produced by expression of an exogenous DNA sequence in a prokaryote or eukaryote. Suitable in principle for producing the protein is any prokaryotic or eukaryotic plasmid vector, bacteriophase vector or yeast plasmid vector. Examples of such vectors are pBR322, pUC18,19, pBluescript, pcDNA3.1 and others.
The natural ligand may already be present in the biological material or the preparation of biological material. However, such a ligand can also be added from outside. For this purpose, the ligand should be present in an amount, concentration and degree of purity such that its binding to the receptor and initiation of the receptor signal is brought about.
Addition of the natural ligand takes place after the biological material or a preparation of the biological material, each of which contain the G protein-coupled receptor GPR451ike/GPR63, is available in a suitable way (preparation/amount).
Such a natural ligand is preferably sphingosine 1-phosphate or lysophosphatidic acid. The natural ligands and, in particular, also the sphingosine 1-phosphate or the lysophosphatidic acid may, for use in the method of the invention, comprise a label which can be detected by a suitable detection method. Such a label is, for example, a radioactive label or a fluorometrically detectable label.
The method of the invention is preferably used in such a way that the identified compounds act agonistically or antagonistically toward the natural ligand.
The invention also relates to a compound which modifies the activity of the G
protein-coupled receptor GPR451ikeIGPR63, where the compound has been identified by a method as described above.
Such a compound is distinguished by having a mass 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 of the invention may 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 comprises a compound which has been identified by a method of the invention, and additionally formulation excipients for a medicament and/or other additives. This medicament is particularly suitable for the treatment of a cardiovascular disorder.
A compound which has been identified by a method of the invention may be used to produce a medicament for treating a cardiovascular disorder.
The invention further relates to the use of a compound which has been identified by a method of the invention for producing a complex of the receptor GPR451ike/GPR63 with this compound. Suitable compounds for producing such a complex are, for example, sphingosine 1-phosphate or lysophosphatidic acid. The invention also relates to a complex of the receptor GPR451ikelGPR63 and sphingosine 1-phosphate and/or lysophosphatidic acid. A complex means in this connection a compound composed of one or more proteins of the receptor in an at least specific binding with sphingosine 1-phosphate and/or lysophosphatidic acid and further lipids, membrane constituents or detergents which are possibly necessary to stabilize the receptor or receptor/ligand complex. The binding is specific if the binding constant is less than or equal to 100 ~NM.
The G protein-coupled receptor GPR451ikeIGPR63 can be cloned and produced by means of a recombinant vector construct.
Recombinant vector constructions can be produced with the assistance of relevant expert knowledge as described, for example, in "F.M. Ausubel et al., Current Protocols in Molecular Biology, Wiley 8~ Sons, New York (ISBN 0-471-50338-X)"
or in "J. Sambrook, E.F. Fritsch, T. Mamiatis, Molecular Cloning, second edition, Cold Spring Harbor Laboratory Pressn (ISBN 0-87969-309-6). This entails a polynucleotide coding for an amino acid sequence as shown in one of the described sequence informations (SEQ ID NO. 1 ) or a polynucleotide sequence as shown in one of the described sequence informations (SEQ ID NO. 2) being incorporated into a basic vector. Basic vector is intended to mean a vector into which a polynucleotide sequence of a polynucleotide can be incorporated by the methods of molecular biology and can be cloned in a microorganism, for example a bacterium, fungus or the cell of a cell culture. The basic vector may consist for example of a plasmid having an antibiotic resistance marker, an origin of replication suitable for replication of the plasmid in bacteria or cell cultures, and a promoter suitable for expression of a protein. The basic vector may also consist for example of a phage vector, a phagemid vector, a phasmid vector, a cosmid vector, a virus vector, a YAC
vector or other vector type. Examples of basic vectors are pUC 18, pUC19, pBluescript, pKS, pSK and others. Incorporation of the polynucleotide which is to be incorporated takes place via suitable restriction cleavage sites using the appropriate restriction enzymes which are commercially available from companies such as BioLabs, Roche Diagnostics, Stratagene and others. Such restriction cleavage sites may be, for example, the recognition sites of the restriction enzymes BamHl, EcoRl, Sall, EcoRV
and others.
The recombinant vector construction consists in a preferred embodiment of an expression vector which can be used in eukaryotes and/or prokaryotes. An expression vector comprises a promoter which can be functionally connected 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 may be inducible for example by tryptophan or in a constitutive activity. Examples of expression vectors are pUC18, pUC19, pBluescript, pcDNA3.1 or others.
Biological material is any material which contains genetic information and can itself reproduce or be reproduced in a biological system. Examples of biological material are cells from human or animal tissues or organs such as, in particular, brain, adipose tissue, lung, heart, liver, kidney, spleen, muscle or others. Examples of biological material are also bacteria or fungi such as, for example, Escherichia coli or Saccharomyces cerevisiae. Biological material also encompasses cells from cell cultures.
Biological material can be obtained in the case of cells from animal or human tissues by biopsy, surgical removal, removal by means of syringes or catheters or comparable techniques. The cells removed in this way can be deep-frozen, worked up or put in cell culture. Bacteria and yeast cells are grown and worked up using conventional techniques of microbiology.
The skilled worker will find appropriate instructions therefor in "Current Protocols in Molecular Biology; ed.: F. M. Ansubel et al., continually revised, 2001 edition, published by John Wiley & Sons".
Biological material may also consist of cells of an animal cell culture.
Examples of such cells are mouse cells, rat cells or hamster cells. The cell culture cells may be primary cell types or established cell lines. Examples of established cell lines are mouse 3T3 cells, CHO cells or Hela cells. The maintenance, culturing and growing of 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 disruption of the biological material and subsequent purification steps. Methods for disruption of the biological material may be in particular repeated freezing and thawing, treatment with ultrasound, the use of a French press, addition of detergents and enzymes or similar.
~ The G protein-coupled receptor GPR451ikeIGPR63 for carrying out the method of the invention can be produced by expression of an exogenous DNA sequence in a prokaryote or eukaryote. Suitable in principle for producing the protein is any prokaryotic or eukaryotic plasmid vector, bacteriophase vector or yeast plasmid vector. Examples of such vectors are pBR322, pUC18,19, pBluescript, pcDNA3.1 and others.
The natural ligand may already be present in the biological material or the preparation of biological material. However, such a ligand can also be added from outside. For this purpose, the ligand should be present in an amount, concentration and degree of purity such that its binding to the receptor and initiation of the receptor signal is brought about.
Addition of the natural ligand takes place after the biological material or a preparation of the biological material, each of which contain the G protein-coupled receptor GPR451ike/GPR63, is available in a suitable way (preparation/amount).
Such a natural ligand is preferably sphingosine 1-phosphate or lysophosphatidic acid. The natural ligands and, in particular, also the sphingosine 1-phosphate or the lysophosphatidic acid may, for use in the method of the invention, comprise a label which can be detected by a suitable detection method. Such a label is, for example, a radioactive label or a fluorometrically detectable label.
The method of the invention is preferably used in such a way that the identified compounds act agonistically or antagonistically toward the natural ligand.
The invention also relates to a compound which modifies the activity of the G
protein-coupled receptor GPR451ikeIGPR63, where the compound has been identified by a method as described above.
Such a compound is distinguished by having a mass 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 of the invention may 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 comprises a compound which has been identified by a method of the invention, and additionally formulation excipients for a medicament and/or other additives. This medicament is particularly suitable for the treatment of a cardiovascular disorder.
A compound which has been identified by a method of the invention may be used to produce a medicament for treating a cardiovascular disorder.
The invention further relates to the use of a compound which has been identified by a method of the invention for producing a complex of the receptor GPR451ike/GPR63 with this compound. Suitable compounds for producing such a complex are, for example, sphingosine 1-phosphate or lysophosphatidic acid. The invention also relates to a complex of the receptor GPR451ikelGPR63 and sphingosine 1-phosphate and/or lysophosphatidic acid. A complex means in this connection a compound composed of one or more proteins of the receptor in an at least specific binding with sphingosine 1-phosphate and/or lysophosphatidic acid and further lipids, membrane constituents or detergents which are possibly necessary to stabilize the receptor or receptor/ligand complex. The binding is specific if the binding constant is less than or equal to 100 ~NM.
The G protein-coupled receptor GPR451ikeIGPR63 can be cloned and produced by means of a recombinant vector construct.
Recombinant vector constructions can be produced with the assistance of relevant expert knowledge as described, for example, in "F.M. Ausubel et al., Current Protocols in Molecular Biology, Wiley 8~ Sons, New York (ISBN 0-471-50338-X)"
or in "J. Sambrook, E.F. Fritsch, T. Mamiatis, Molecular Cloning, second edition, Cold Spring Harbor Laboratory Pressn (ISBN 0-87969-309-6). This entails a polynucleotide coding for an amino acid sequence as shown in one of the described sequence informations (SEQ ID NO. 1 ) or a polynucleotide sequence as shown in one of the described sequence informations (SEQ ID NO. 2) being incorporated into a basic vector. Basic vector is intended to mean a vector into which a polynucleotide sequence of a polynucleotide can be incorporated by the methods of molecular biology and can be cloned in a microorganism, for example a bacterium, fungus or the cell of a cell culture. The basic vector may consist for example of a plasmid having an antibiotic resistance marker, an origin of replication suitable for replication of the plasmid in bacteria or cell cultures, and a promoter suitable for expression of a protein. The basic vector may also consist for example of a phage vector, a phagemid vector, a phasmid vector, a cosmid vector, a virus vector, a YAC
vector or other vector type. Examples of basic vectors are pUC 18, pUC19, pBluescript, pKS, pSK and others. Incorporation of the polynucleotide which is to be incorporated takes place via suitable restriction cleavage sites using the appropriate restriction enzymes which are commercially available from companies such as BioLabs, Roche Diagnostics, Stratagene and others. Such restriction cleavage sites may be, for example, the recognition sites of the restriction enzymes BamHl, EcoRl, Sall, EcoRV
and others.
The recombinant vector construction consists in a preferred embodiment of an expression vector which can be used in eukaryotes and/or prokaryotes. An expression vector comprises a promoter which can be functionally connected 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 may be inducible for example by tryptophan or in a constitutive activity. Examples of expression vectors are pUC18, pUC19, pBluescript, pcDNA3.1 or others.
Biological material is any material which contains genetic information and can itself reproduce or be reproduced in a biological system. Examples of biological material are cells from human or animal tissues or organs such as, in particular, brain, adipose tissue, lung, heart, liver, kidney, spleen, muscle or others. Examples of biological material are also bacteria or fungi such as, for example, Escherichia coli or Saccharomyces cerevisiae. Biological material also encompasses cells from cell cultures.
Biological material can be obtained in the case of cells from animal or human tissues by biopsy, surgical removal, removal by means of syringes or catheters or comparable techniques. The cells removed in this way can be deep-frozen, worked up or put in cell culture. Bacteria and yeast cells are grown and worked up using conventional techniques of microbiology.
The skilled worker will find appropriate instructions therefor in "Current Protocols in Molecular Biology; ed.: F. M. Ansubel et al., continually revised, 2001 edition, published by John Wiley & Sons".
Biological material may also consist of cells of an animal cell culture.
Examples of such cells are mouse cells, rat cells or hamster cells. The cell culture cells may be primary cell types or established cell lines. Examples of established cell lines are mouse 3T3 cells, CHO cells or Hela cells. The maintenance, culturing and growing of 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 disruption of the biological material and subsequent purification steps. Methods for disruption of the biological material may be in particular repeated freezing and thawing, treatment with ultrasound, the use of a French press, addition of detergents and enzymes or similar.
Subsequent purification steps consist, for example, of differential centrifugation, precipitation with ammonium sulfate or organic solvents, 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, and in particular also for detailed instructions for the purification of proteins, detailed instructions are available to the skilled worker in textbooks such as, in particular, in "Current Protocols in Protein Science, ed.: J.E.
Coligan et al., continually revised, 2001 edition, published by John Wiley &
Sons.
The biological material or the preparation of biological material can be brought into contact with a chemical compound in conventional laboratory vessels such as, for example, Eppendorf vessels, centrifuge tubes or glass flasks. The underlying aqueous medium comprises, for example, buffer substances, nutrient constituents, singly charged or doubly charged ions such as Na+, K+, Ca2+, CI-, S042-, PO32-or others, also proteins, glycerol or others. Particular constant conditions such as, in particular, the temperature, the pH, the ionic conditions, the concentration of a protein, the volume or other factors may be advantageous for the bringing into contact. This is achieved by, for example, carrying out the bringing into contact in incubation apparatuses kept at a constant temperature, in the presence of a buffer or with the previously accurately weighed amounts of the ions or proteins. The aqueous solvent may in particular also comprise a certain proportion of an organic solvent such as dimethyl sulfoxide, methanol or ethanol. The content of such a solvent is, however, preferably not more than 10% by volume of the mixture.
The provision of a chemical compound takes place for example by chemical synthesis. The skilled worker is familiar with standard methods of synthesis.
The chemical compound may be part of a collection of chemical compounds like those produced by storage and cataloging of the chemical compounds from closed synthesis programs (called chemical libraries). The compound may in other cases have been produced by a microorganism, in particular a bacterium, but also by a fungus or a plant (natural product).
Suitable pharmaceutical compounds can be administered as medicaments in oral, peroral, topical, parenteral or rectal form. The mode of administration which is most suitable depends in each individual case on the nature and severity of the condition to be treated and on the type of compound used in each case. A suitable active 5 ingredient concentration is about 1 % to 35%, preferably about 3% to 15%.
Examples Cloning of the human GPR451ike/GPR63 receptor The sequence of the human GPR451ike receptor is accessible to the public under numbers AF 31765 and AB 030566 at Genbank and EMBL. The human gene contains no introns. The receptor has therefore been amplified starting from human genomic DNA using a polymerise chain reaction (PCR). The PCR reaction conditions were as follows:
Initially incubation at 94°C for 10 min, then 35 cycles of incubation at 94°C in each case for 1 min per cycle, then incubation at 60°C for 1 min and finally incubation at 72°C for 2 min. The reaction was in this case carried out using the GC-melt Kit from Clontech. The primers used as shown in SEQ ID No. 3 and 4 were designed so that they contained a Hindlll cleavage site (SEQ ID No. 3) and an EcoRl cleavage site (SEQ ID No. 4). The PCR fragment with a length of 1 260 base pairs which was formed by the PCR described above was incorporated with the aid of the Hindlll and EcoRl cleavage sites into the eukaryotic expression vector pCDNA 3.1 (+). This vector is commercially obtainable for example from Invitrogen, Carlsbad, California.
In an extraction of RNA; PCR using reverse transcriptase (RT-PCR) RNA was isolated from various cell lines.
In the present case, TRlzol reagent from Gibco BRL was used for this.
The RNA was isolated from the following cell lines: HUVECS (human umbilical vein endothelial cells). HPAEC (human pulmonary artery endothelial cells), Hek 293 (human embryonic kidney cells), HCASMC (human coronary artery smooth muscle cells), HCAEC (human coronary artery endothelial cells), HMVEC-L (human microvascular endothelial cells of the lung), HPASMC (human pulmonary artery smooth muscle cells), HAOSMC (human aortic smooth muscle cells).
The cells were harvested shortly before confluence was reached in a tissue culture bottle. RNA was isolated from the cells in accordance with the instructions of the manufacturer of TRI zol reagent. The RNA was tested for the absence of genomic DNA. About 5 Ng of this RNA was used to carry out a reverse transcriptase reaction using MMLV (Moloney marine leukemia virus) reverse transcriptase and the RT-PCR
kit from Stratagene. The RT-PCR was carried out in a volume of 50 NI, the reaction being carried out by incubation initially at 65°C for 5 min, then at room temperature for 15 min, then at 37°C for 1 hour and then at 90°C for 5 min and finally by cooling in ice.
The cDNA preparations from these reverse transcriptions were used for the subsequent PCR reactions. Where available, cDNA obtainable commercially was employed.
About 5 Ng of cDNA were used for a PCR. The reaction itself is carried out with an Amplitaq Gold Polyme~ase Kit from Perkin Elmer. The reaction conditions for the cycles are as follows:
incubation at 95°C for 12 min, then 35 cycles incubating at 94°C
in each case initially for 1 min and then incubating at 72°C for 1 min for each cycle and then, after completing the 35 cycles, incubating at 72°C for 10 min and finally cooling on ice.
The primers used for this purpose were the two following DNA sequences:
5'-CCC ACT GGT TTG AGT TCC TTG ACC-3' (SEQ ID No. 5; "forward"), 5'-GGT AGC CTG GAT TGG TTG TGT ACC-3' (SEQ ID No. 6; "reverse") A product 561 base-pairs long resulted.
Quantitative RT-PCR in real time using Taqman The quantitative PCR analysis was carried out by means of fluorescence resonance energy transfer (FRET). The skilled worker is familiar with this system also under the name Taqman PCR. A kit commercially available from Perkin Elmer was used to carry out the TaqMan reaction. The Taqman sample consists of a single-stranded oligonucleotide which is labeled with 2 different fluorophores. The fluorophore at the 3' end (acceptor) acts as "quencher" (i.e. with attenuating effect) of the fluorophore at the 5' end (donor). The Taq DNA polymerise liberates the fluorophore at the 5' end through its 5'-exonuclease activity during the chain-extension reaction. Since the emission of the fluorophore is now no longer quenched, it can be measured by a fluorimeter. The amount of fluorescence found is directly proportional to the amount of the PCR product which accumulates during the amplification. Care must be taken that the melting temperature of the Taqman oligo is higher than that of the primer for the amplification by Taq polymerise.
The fluorophores used in the present case were FAM (6-carboxyfluorescein) as donor for the 5' end and TAMRA (5-carboxytetramethylrhodamine) as acceptor/quencher for the 3' end.
The cDNA was produced by using commercially available RNA, for example from Clontech, Heidelberg.
5 Ng of total RNA were mixed with 2.5 NI (5 mg/Nl) of hexamer primers of random sequence. Such hexamer primers are available from various companies. In the present case, they were from Invitrogen (Life Technologies), Karlsruhe.
Total RNA and hexamer primers were first heated at 70°C for 10 min and cooled on ice. Then 4 Nl of 5 x buffer (first stand buffer), 2 NI of 0.1 nM DTT
(dithiothreitol), 1 p1 of 10 mM dNTP and 1 Nl of water were added and, after careful mixing, incubated at 37°C for 2 minutes. 5 NI of reverse transcriptase were then added, and the mixture was incubated at 37°C for 60 min. The reaction was stopped by adding 1 NI of 2.5 mM EDTA and heating at 65°C for 10 min.
The cDNA samples produced in this way served as templates for the subsequent quantitative PCR.
In each case 50 ng/NI, 25 ng/NI, 10 ng/pl, 5 ng/NI, 2.5 ng/pl, 1.25 ng/Nl and 0.625 ng/pl final concentration of each reverse-transcribed cDNA template were investigated.
The reaction took place in a total volume of 50 NI. The reaction mixture contains dNTP and buffer in the usual concentrations, and Taq polymerase.
The final concentration of the primers was 900 mM in each case.
A comparison was undertaken with primers for the human beta actin gene in human brain cDNA.
The determinations were each carried out in duplicate in different batches.
The standard values for the human actin gene were used as internal controls to standardize the samples in the determination of the GPR451ike/GPR63 gene expression to be found in the various RNAs. The expression was expressed as a ratio to a previously defined reference tissue. The reference defined in each case was the cerebellum for the central nervous system and the brain for the peripheral tissue.
The primers used for the amplification reaction in the first determination had the following nucleotide sequence 5'- CTC AAC ACC CTT CGC CAC A-3' (SEQ ID No. 7) 5'-GGCC TGG CTG AGG CAT ATAC-3' (SEQ ID No. 8).
The Taqman oligo sample for this had the nucleotide sequence:
5'-TGC CTT GAG GAT CCA TAG CTA CCC TGA A-3' (SEQ iD No. 9).
The amplification primers in the second determination had the following nucleotide sequence:
5'-TGCC TGG ACA TGA TGC CTA A-3' (SEQ ID No. 10) 5'-TCC GTC GCT TTG TGT GAC C-3' (SEQ ID No. 11).
The corresponding Taqman sample for this had the nucleotide sequence:
5'-TCC TTC AAC TTT TTG CCG CAG CTC C-3' (SEQ ID No. 12).
Northern blotting Northern blots with RNA from various human tissues were purchased from Ciontech, Palo Alto. Such Northern blots can also be obtained in the same quality from other suppliers. The nucleotide sequence of the GPR451ike/GPR63 receptor was cut out using EcoRl and Hindlll and expression plasma drive from pcDNA 3.1 and was fractionated on an agarose gef and the sequence 1 260 base pairs long was isolated and then radiolabeled with 3ZP-dCTP. The Northern blots with the RNA from various tissues were hybridized under stringent conditions. The radioactivity was detected using a film. For internal comparison, the same Northern blots were washed until none of the previously hybridized DNA molecules with the coding sequence for the GPR451ike/GPR63 receptor were detectable any longer. These washed Northern blots were again hybridized using a beta-actin cDNA sample.
Detection of GPR451ike/GPR63 receptor activity in transfected cell cultures CHO-K1 cells were cultivated in basal Iscove's medium with further additions.
Iscove's medium is commercially available for example from Biochrom. The composition was described for the first time in "iscove, N.N., Melchers, F., Journal of Experimental Medicine 147, 923-933 (1978). 10 % fetal bovine serum, 10 000 U/ml-10 000 Ng/ml penicillin-streptomycin, gentamycin, and 2 mM L-glutamine were used as further additions to lscove's medium. The cells were incubated at 37°C with a 5%
C02 atmosphere.
About 2 x 10$ CHO-K1 cells were seeded in 35 mm dishes for the transient transfection. After further incubation for about 24 hours and with the cells at about 50-80% confluence, the cells were transiently transfected with 1 pg of the plasmid DNA construct with the assistance of Lipofectamine (Gibco).
FLIPR assay {fluorometric imaging plate reader assay) About 16 to 18 hours after the transfection, the CHO cells were put in an amount of 5 about 80 000 cells per well in 96-well plates and incubated further for about 18-24 hours. 95 NI of HBSS (Hank's buffered saline solution) with 20 mM HEPES, 2.5 mM
probenecid {4-[(dipropylamino)sulfonyl]benzoic acid) and 4 NM dye FIuo4 were added to the cells.
10 The cells were incubated in 5% C02 for 1 hour and washed three times with PBS
(phosphate buffered saline) which contained 1 mM MgCl2, 1 mM MEDTA, 0.4 rnglml FAF-BSA (fatty acid free bovine serum albumin) and 2.5 mM protenecid.
After the last washing step, 100 NI were left on the cells in each well. The lipids to be 15 tested were available as 2 mM stock solution in DMSO (dimethylsulfoxide). A
60 pM
solution was kept ready on a 96-well microtiter plate. The stock solution was diluted in PBS. 50 NI portions of this 6 pM solution were transferred into each well of the microtiter plate containing 100 NI of PBS and the cells. A 20 NM final concentration of the lipids to be tested was obtained in this way.
The fluorescence was measured by the instrument (FLIPR, Molecular Devices) for 1 min in intervals lasting 1 sec and for a further 2 min in 3-second intervals.
Cell growth assay CHO cells were put in an amount of 8 x 104 cells into 35 mm plates. After 32 hours, the cells were transfected with 1 Ng of the plasmid construct using Lipofectamine (Gibco). After a further 13 hours (time 0), the cells were washed with PBS and incubated in Iscove's medium which contained 10% dialyzed fetal calf serum for hours (time 48), specifically in the presence or absence of 1 pM S1 P
(sphingosine 1-phosphate).
The number of cells at time 0 and time 48 was determined by counting. This was done by first treating the cells with trypsin and then suspending them in 1 ml of Iscove's. An amount of 100 ml of these cells was diluted in 10 mf of buffer and counted in a cell counter, e.g, in the Casy Cellcounter TT (Cell Counter and Analysesystem, Scharfe, Reutlingen).
Specificity of expression of the GPR451ike/GPR63 receptor in various human tissues The expression was investigated using the RT-PCR. Specific transcripts of the GPR451ike/GPR63 receptor were detectable in the aorta, heart, left ventricle, fetal hearts, brain and kidney. Only a weak band was obtainable in tissues from the left atrium. No signal was detectable in lung tissue.
Expression of the GPR451ike/GPR63 receptor in various human cell lines HUVECS (human umbilical vein endothelial cells), HCAEC (human coronary artery endothelial cells), MHVEC-L (human microvascular endothelial cells from lung), HPAEC (human coronary artery smooth muscle cells), HPASMC (human pulmonary artery smooth muscle cells), HAOSMC (human aortic smooth muscle cells) were investigated.
Transcripts of the GPR451ike/GPR63 receptor were detectable in all cell lines.
GPR451ike/GPR63 receptor expression in peripheral human tissue Expression of the receptor was determined using the TaqMan RT-PCR analysis semiquantitatively in relation to the expression in the brain.
An internal comparison took place in relation to the f3-actin RNA. Each measurement was determined twice. The following table shows the RNA expression as a multiple of the expression in the brain.
Table:
Expression of the GPR451ike/GPR63 receptor in various tissues in relation to the brain Expression relative to brain Brain 1 Heart 0.1 Kidney 0.2 Liver 0.01 Lung 0.05 Spleen 0.2 Thymus 0.6 Skeletal muscle 0.05 Pancreas 0.15 Small intestine 0.8 Stomach 0.7 .
Relatively strong expression was found in thymus, small intestine and stomach, while almost no expression was detectable in liver, kidney and skeletal muscle.
GPR451ike/GPR63 receptor expression in various parts of the brain.
Expression of the receptor was determined using the TaqMan RT-PCR
semiquantitatively in relation to expression in the cerebellum. The internal comparison was in relation to the f3-actin RNA. Each measurement was determined twice. The following table shows the RNA to the cerebellum.
Table:
Expression of the GPR451ike/GPR63 receptor in various parts of the brain in relation to the cerebellum Expression relative to the cerebellum Cerebellum 1 Whole brain 1.2 Corpus callosum 0.2 Caudatus 1.5 Thalamus 2.1 Amygdala 1.2 The strongest expression by comparison is observed in the thalamus.
Stimulation of the intracellular Ca2+ release by the GPR451ike/GPR63 receptor 209 different bioactive lipids from a substance library were added, each in a final concentration of 1 NM, to CHO cells which expressed the GPR451ikeIGPR63 receptor and the G Protein a subunit delta 6qi4mgr (= i49i4). About 30 compounds were tested. Only S1 P (sphingosine 1-phosphate) and DHS1 P
(dihydrosphingosine 1-phosphate) led to a measurable Ca2+ influx. S1 P and DHS1 P are naturally occurring lipid-like substances which also appear as natural ligands of other GPCRs.
The a subunit delta 6qi4mgr (= i4qi4) has a broad specificity in relation to different GPCRs.
Induction of cell growth by GPR451ike/GPR63 receptors.
An amount of 8 x 104 CHO cells was seeded onto 35 mm plates. The cells were transfected after 32 hours with 1 Ng of DNA of the vector construction. After hours, the cells were washed once with PBS and incubated in Iscove's medium containing 10% dialyzed fetal calf serum in the presence or absence of 1 NM S1 P for a further 48 hours. It was possible to show that test mixtures which contained contained about 20% more cells in the average of 2 tests done independently of one another.
SEQUENCE LISTING
<110> Aventis Pharma Deutschland GmbH
< 12 0 > Method for identif)ring agonists or antagonists of the GPR45 Iike/GP63 receptor <130> DEAV2002J0033 <140>
<141>
<160> 12 c170> PatentIn Ver. 2.1 <210> 1 c211> 1260 <212> DNA
<213> Homo sapiens <400> 1 atggtcttct cggcagtgtt gactgcgttc cataccggga catccaacac aacatttgtc 60 gtgtatgaaa acacctacat gaatattaca ctccctccac cattccagca tcctgacctc 120 agtccattgc ttagatatag ttttgaaacc atggctccca ctggtttgag ttccttgacc 180 gtgaatagta cagctgtgcc cacaacacca gcagcattta agagcctaaa cttgcctctt 240 cagatcaccc tttctgctat aatgatattc attctgtttg tgtcttttct tgggaacttg 300 gttgtttgcc tcatggttta ccaaaaagct gccatgaggt ctgcaattaa catcctcctt 360 gccagcctag cttttgcaga catgttgctt gcagtgctga acatgccctt tgccctggta 420 actattctta ctacccgatg gatttttggg aaattcttct gtagggtatc tgctatgttt 480 sttctggttat ttgtgataga aggagtagcc atcctgctca tcattagcat agataggttc 540 cttattatag tccagaggca ggataagcta aacccatata gagctaaggt tctgattgca 600 gtttcttggg caacttcctt ttgtgtagct tttcctttag ccgtaggaaa ccccgacctg 660 cagatacctt cccgagctcc ccagtgtgtg tttgggtaca caaccaatcc aggctaccag 720 gcttatgtga ttttgatttc_ tctcatttct ttcttcatac ccttcctggt aatactgtac 780 tcatttatgg gcatactcaa cacccttcgg cacaatgcct tgaggatcca tagctaccct 840 gaaggtatat gcctcagcca ggccagcaaa ctgggtctca tgagtctgca gagacctttc 900 cagatgagca ttgacatggg ctttaaaaca cgtgccttca ccactatttt gattctcttt 960 gctgtcttca ttgtctgctg ggccccattc accacttaca gccttgtggc aacattcagt 1020 aagcactttt actatcagca caactttttt gagattagca cctggctact gtggctctgc 1080 tacctcaagt ctgcattgaa tccgctgatc tactactgga ggattaagaa attccatgat 1140 gcttgcctgg acatgatgcc taagtccttc aagtttttgc cgcagctccc tggtcacaca 1200 aagcgacgga tacgtcctag tgctgtctat gtgtgtgggg aacatcggac ggtggtgtga 1260 <210> 2 <211> 4i9 <212> PRT
<213> Homo Sapiens <400> 2 Met Val Phe Ser Ala Val Leu Thr Ala Phe His Thr Gly Thr Ser Asn Thr Thr Phe Val Val Tyr Glu Asn Thr Tyr Met Asn Ile Thr Leu Pro Pro Pro Phe Gln His Pro Asp Leu Sez Pro Leu Leu Arg Tyr Ser Phe Glu Thr Met Ala Pro Thr Gly Leu Ser Ser Leu Thr Val Asn Ser Thr Ala Val Pro Thr Thr Pro Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu 65 70 T5 . 80 Gln Ile Thr Leu Ser Ala Ile Met Ile Phe Ile Leu Phe Val Ser Phe Leu Gly Asn Leu Val Val ors Leu Met Val Tyr Gln Lys Ala Ala Met Arg Ser Ala Ile Asn Ile Leu Leu Ala Ser Leu Ala Phe Ala Asp Met Leu Leu Ala Val Leu Asn Met Pro Phe Ala Leu Val Thr Ile Leu Thr Thr Arg Trp Ile Phe Gly Lys Phe Phe Cys Arg Val Ser Ala Met Phe Phe Trp Leu Phe Val Ile Glu Gly Val Ala Ile Leu Leu Ile Ile Ser Ile Asg Arg Phe Leu Ile Ile Val Gln Azg Gln Asp Lys Leu Asn Pro Tyr Arg Ala Lys Val Leu Ile Ala Val Ser Trp Ala Thr Ser Phe Cys Val Ala Phe Pro Leu Ala Val Gly Asn Pro Asp Leu Gln Ile Pro Ser Arg Ala Pro Gln Cys Val Phe Gly Tyr Thr Thr Asn Pro Gly Tyr Gln Ala Tyr Val Ile Leu Ile Ser Leu Ile Ser Phe Phe Ile Pro Phe Leu Val Ile Leu Tyr Ser Phe Met Gly Ile Leu Asn Thr Leu Arg His Asn 2fi0 2fi5 2?0 Ala Leu Arg Ile His Ser Tyr Pro Glu Gly Ile Cys Leu Ser Gln Ala Ser Lys Leu Gly Leu Met Ser Leu Gln Arg Pra Phe Gln Met Ser Ile 290 295 300 .
Asp Met Gly Phe Lys Thr Arg Ala Phe Thr Thr Ile Leu Ile Leu Phe Ala Val Phe Ile Val Cys Trp Ala Pro Phe Thr Thr Tyr Ser Leu Val Ala Thr Phe Ser Lys His Phe Tyr Tyr Gln His Asn Phe Phe Glu Ile Ser Thr Trp Leu Leu Trp Leu Cys Tyr Leu Lys Ser Ala Leu Aan Pro Leu Ile Tyr Tyr Trp Arg Ile Lys Lys Phe His Asp Ala Cys Leu Asp 370 375 380 .
Met Met Pro Lys Ser Phe Lys Phe Leu Pro Gln Leu Pro Gly His Thr Lys Arg Arg Ile Arg Pro 5er Ala Val Tyr Val Cys Gly Glu His Arg Thr Val Val <210> 3 <211> 42 <212> DNA
<213> Homo Sapiens <400> 3 ccgccgaagc ttgccatggt cttctcggca gtgttgactg cg 42 <210> 4 <211> 36 <212> DNA
<213> Homo Sapiens c4D0> 9 gccggcgaat tctcacacca ccgtccgatg ttcccc 36 <210>5 <211>24 <212>DNA
<213>Homo Sapiens <400> 5 cccactggtt tgagttcctt gacc 2g <210> 6 <211> 24 <212> DNA
<213> Homo Sapiens c400> 6 ggtagcctgg attggttgtg tacc 2q <210> 7 <211> 18 <212> DNA
<213> Homo Sapiens <400> 7 ctcaacaccc ttcggcac ig <210> 8 <211> 20 <212> DNA
<213> Homo Sapiens <400> 8 ggcctggctg aggcatatac 2D
<210> 9 <211> 28 <212> DNA
<213> Homo Sapiens <400> 9 tgccttgagg atccatagct accctgaa 2B
<210> 10 <211> 20 <212> DNA
<213> Homo sapiens <400> 10 tgcctggaca tgatgcctaa 2 <210> 11 <211> 19 <212> DNA
c213> Homo sapiens <400> 11 tccgtcgctt tgtgtgacc 19 c210> 12 c211> 25 <212> DNA
<213> Homo sapiens c400> 12 tccttcaagt ttttgccgca gctcc 25
Coligan et al., continually revised, 2001 edition, published by John Wiley &
Sons.
The biological material or the preparation of biological material can be brought into contact with a chemical compound in conventional laboratory vessels such as, for example, Eppendorf vessels, centrifuge tubes or glass flasks. The underlying aqueous medium comprises, for example, buffer substances, nutrient constituents, singly charged or doubly charged ions such as Na+, K+, Ca2+, CI-, S042-, PO32-or others, also proteins, glycerol or others. Particular constant conditions such as, in particular, the temperature, the pH, the ionic conditions, the concentration of a protein, the volume or other factors may be advantageous for the bringing into contact. This is achieved by, for example, carrying out the bringing into contact in incubation apparatuses kept at a constant temperature, in the presence of a buffer or with the previously accurately weighed amounts of the ions or proteins. The aqueous solvent may in particular also comprise a certain proportion of an organic solvent such as dimethyl sulfoxide, methanol or ethanol. The content of such a solvent is, however, preferably not more than 10% by volume of the mixture.
The provision of a chemical compound takes place for example by chemical synthesis. The skilled worker is familiar with standard methods of synthesis.
The chemical compound may be part of a collection of chemical compounds like those produced by storage and cataloging of the chemical compounds from closed synthesis programs (called chemical libraries). The compound may in other cases have been produced by a microorganism, in particular a bacterium, but also by a fungus or a plant (natural product).
Suitable pharmaceutical compounds can be administered as medicaments in oral, peroral, topical, parenteral or rectal form. The mode of administration which is most suitable depends in each individual case on the nature and severity of the condition to be treated and on the type of compound used in each case. A suitable active 5 ingredient concentration is about 1 % to 35%, preferably about 3% to 15%.
Examples Cloning of the human GPR451ike/GPR63 receptor The sequence of the human GPR451ike receptor is accessible to the public under numbers AF 31765 and AB 030566 at Genbank and EMBL. The human gene contains no introns. The receptor has therefore been amplified starting from human genomic DNA using a polymerise chain reaction (PCR). The PCR reaction conditions were as follows:
Initially incubation at 94°C for 10 min, then 35 cycles of incubation at 94°C in each case for 1 min per cycle, then incubation at 60°C for 1 min and finally incubation at 72°C for 2 min. The reaction was in this case carried out using the GC-melt Kit from Clontech. The primers used as shown in SEQ ID No. 3 and 4 were designed so that they contained a Hindlll cleavage site (SEQ ID No. 3) and an EcoRl cleavage site (SEQ ID No. 4). The PCR fragment with a length of 1 260 base pairs which was formed by the PCR described above was incorporated with the aid of the Hindlll and EcoRl cleavage sites into the eukaryotic expression vector pCDNA 3.1 (+). This vector is commercially obtainable for example from Invitrogen, Carlsbad, California.
In an extraction of RNA; PCR using reverse transcriptase (RT-PCR) RNA was isolated from various cell lines.
In the present case, TRlzol reagent from Gibco BRL was used for this.
The RNA was isolated from the following cell lines: HUVECS (human umbilical vein endothelial cells). HPAEC (human pulmonary artery endothelial cells), Hek 293 (human embryonic kidney cells), HCASMC (human coronary artery smooth muscle cells), HCAEC (human coronary artery endothelial cells), HMVEC-L (human microvascular endothelial cells of the lung), HPASMC (human pulmonary artery smooth muscle cells), HAOSMC (human aortic smooth muscle cells).
The cells were harvested shortly before confluence was reached in a tissue culture bottle. RNA was isolated from the cells in accordance with the instructions of the manufacturer of TRI zol reagent. The RNA was tested for the absence of genomic DNA. About 5 Ng of this RNA was used to carry out a reverse transcriptase reaction using MMLV (Moloney marine leukemia virus) reverse transcriptase and the RT-PCR
kit from Stratagene. The RT-PCR was carried out in a volume of 50 NI, the reaction being carried out by incubation initially at 65°C for 5 min, then at room temperature for 15 min, then at 37°C for 1 hour and then at 90°C for 5 min and finally by cooling in ice.
The cDNA preparations from these reverse transcriptions were used for the subsequent PCR reactions. Where available, cDNA obtainable commercially was employed.
About 5 Ng of cDNA were used for a PCR. The reaction itself is carried out with an Amplitaq Gold Polyme~ase Kit from Perkin Elmer. The reaction conditions for the cycles are as follows:
incubation at 95°C for 12 min, then 35 cycles incubating at 94°C
in each case initially for 1 min and then incubating at 72°C for 1 min for each cycle and then, after completing the 35 cycles, incubating at 72°C for 10 min and finally cooling on ice.
The primers used for this purpose were the two following DNA sequences:
5'-CCC ACT GGT TTG AGT TCC TTG ACC-3' (SEQ ID No. 5; "forward"), 5'-GGT AGC CTG GAT TGG TTG TGT ACC-3' (SEQ ID No. 6; "reverse") A product 561 base-pairs long resulted.
Quantitative RT-PCR in real time using Taqman The quantitative PCR analysis was carried out by means of fluorescence resonance energy transfer (FRET). The skilled worker is familiar with this system also under the name Taqman PCR. A kit commercially available from Perkin Elmer was used to carry out the TaqMan reaction. The Taqman sample consists of a single-stranded oligonucleotide which is labeled with 2 different fluorophores. The fluorophore at the 3' end (acceptor) acts as "quencher" (i.e. with attenuating effect) of the fluorophore at the 5' end (donor). The Taq DNA polymerise liberates the fluorophore at the 5' end through its 5'-exonuclease activity during the chain-extension reaction. Since the emission of the fluorophore is now no longer quenched, it can be measured by a fluorimeter. The amount of fluorescence found is directly proportional to the amount of the PCR product which accumulates during the amplification. Care must be taken that the melting temperature of the Taqman oligo is higher than that of the primer for the amplification by Taq polymerise.
The fluorophores used in the present case were FAM (6-carboxyfluorescein) as donor for the 5' end and TAMRA (5-carboxytetramethylrhodamine) as acceptor/quencher for the 3' end.
The cDNA was produced by using commercially available RNA, for example from Clontech, Heidelberg.
5 Ng of total RNA were mixed with 2.5 NI (5 mg/Nl) of hexamer primers of random sequence. Such hexamer primers are available from various companies. In the present case, they were from Invitrogen (Life Technologies), Karlsruhe.
Total RNA and hexamer primers were first heated at 70°C for 10 min and cooled on ice. Then 4 Nl of 5 x buffer (first stand buffer), 2 NI of 0.1 nM DTT
(dithiothreitol), 1 p1 of 10 mM dNTP and 1 Nl of water were added and, after careful mixing, incubated at 37°C for 2 minutes. 5 NI of reverse transcriptase were then added, and the mixture was incubated at 37°C for 60 min. The reaction was stopped by adding 1 NI of 2.5 mM EDTA and heating at 65°C for 10 min.
The cDNA samples produced in this way served as templates for the subsequent quantitative PCR.
In each case 50 ng/NI, 25 ng/NI, 10 ng/pl, 5 ng/NI, 2.5 ng/pl, 1.25 ng/Nl and 0.625 ng/pl final concentration of each reverse-transcribed cDNA template were investigated.
The reaction took place in a total volume of 50 NI. The reaction mixture contains dNTP and buffer in the usual concentrations, and Taq polymerase.
The final concentration of the primers was 900 mM in each case.
A comparison was undertaken with primers for the human beta actin gene in human brain cDNA.
The determinations were each carried out in duplicate in different batches.
The standard values for the human actin gene were used as internal controls to standardize the samples in the determination of the GPR451ike/GPR63 gene expression to be found in the various RNAs. The expression was expressed as a ratio to a previously defined reference tissue. The reference defined in each case was the cerebellum for the central nervous system and the brain for the peripheral tissue.
The primers used for the amplification reaction in the first determination had the following nucleotide sequence 5'- CTC AAC ACC CTT CGC CAC A-3' (SEQ ID No. 7) 5'-GGCC TGG CTG AGG CAT ATAC-3' (SEQ ID No. 8).
The Taqman oligo sample for this had the nucleotide sequence:
5'-TGC CTT GAG GAT CCA TAG CTA CCC TGA A-3' (SEQ iD No. 9).
The amplification primers in the second determination had the following nucleotide sequence:
5'-TGCC TGG ACA TGA TGC CTA A-3' (SEQ ID No. 10) 5'-TCC GTC GCT TTG TGT GAC C-3' (SEQ ID No. 11).
The corresponding Taqman sample for this had the nucleotide sequence:
5'-TCC TTC AAC TTT TTG CCG CAG CTC C-3' (SEQ ID No. 12).
Northern blotting Northern blots with RNA from various human tissues were purchased from Ciontech, Palo Alto. Such Northern blots can also be obtained in the same quality from other suppliers. The nucleotide sequence of the GPR451ike/GPR63 receptor was cut out using EcoRl and Hindlll and expression plasma drive from pcDNA 3.1 and was fractionated on an agarose gef and the sequence 1 260 base pairs long was isolated and then radiolabeled with 3ZP-dCTP. The Northern blots with the RNA from various tissues were hybridized under stringent conditions. The radioactivity was detected using a film. For internal comparison, the same Northern blots were washed until none of the previously hybridized DNA molecules with the coding sequence for the GPR451ike/GPR63 receptor were detectable any longer. These washed Northern blots were again hybridized using a beta-actin cDNA sample.
Detection of GPR451ike/GPR63 receptor activity in transfected cell cultures CHO-K1 cells were cultivated in basal Iscove's medium with further additions.
Iscove's medium is commercially available for example from Biochrom. The composition was described for the first time in "iscove, N.N., Melchers, F., Journal of Experimental Medicine 147, 923-933 (1978). 10 % fetal bovine serum, 10 000 U/ml-10 000 Ng/ml penicillin-streptomycin, gentamycin, and 2 mM L-glutamine were used as further additions to lscove's medium. The cells were incubated at 37°C with a 5%
C02 atmosphere.
About 2 x 10$ CHO-K1 cells were seeded in 35 mm dishes for the transient transfection. After further incubation for about 24 hours and with the cells at about 50-80% confluence, the cells were transiently transfected with 1 pg of the plasmid DNA construct with the assistance of Lipofectamine (Gibco).
FLIPR assay {fluorometric imaging plate reader assay) About 16 to 18 hours after the transfection, the CHO cells were put in an amount of 5 about 80 000 cells per well in 96-well plates and incubated further for about 18-24 hours. 95 NI of HBSS (Hank's buffered saline solution) with 20 mM HEPES, 2.5 mM
probenecid {4-[(dipropylamino)sulfonyl]benzoic acid) and 4 NM dye FIuo4 were added to the cells.
10 The cells were incubated in 5% C02 for 1 hour and washed three times with PBS
(phosphate buffered saline) which contained 1 mM MgCl2, 1 mM MEDTA, 0.4 rnglml FAF-BSA (fatty acid free bovine serum albumin) and 2.5 mM protenecid.
After the last washing step, 100 NI were left on the cells in each well. The lipids to be 15 tested were available as 2 mM stock solution in DMSO (dimethylsulfoxide). A
60 pM
solution was kept ready on a 96-well microtiter plate. The stock solution was diluted in PBS. 50 NI portions of this 6 pM solution were transferred into each well of the microtiter plate containing 100 NI of PBS and the cells. A 20 NM final concentration of the lipids to be tested was obtained in this way.
The fluorescence was measured by the instrument (FLIPR, Molecular Devices) for 1 min in intervals lasting 1 sec and for a further 2 min in 3-second intervals.
Cell growth assay CHO cells were put in an amount of 8 x 104 cells into 35 mm plates. After 32 hours, the cells were transfected with 1 Ng of the plasmid construct using Lipofectamine (Gibco). After a further 13 hours (time 0), the cells were washed with PBS and incubated in Iscove's medium which contained 10% dialyzed fetal calf serum for hours (time 48), specifically in the presence or absence of 1 pM S1 P
(sphingosine 1-phosphate).
The number of cells at time 0 and time 48 was determined by counting. This was done by first treating the cells with trypsin and then suspending them in 1 ml of Iscove's. An amount of 100 ml of these cells was diluted in 10 mf of buffer and counted in a cell counter, e.g, in the Casy Cellcounter TT (Cell Counter and Analysesystem, Scharfe, Reutlingen).
Specificity of expression of the GPR451ike/GPR63 receptor in various human tissues The expression was investigated using the RT-PCR. Specific transcripts of the GPR451ike/GPR63 receptor were detectable in the aorta, heart, left ventricle, fetal hearts, brain and kidney. Only a weak band was obtainable in tissues from the left atrium. No signal was detectable in lung tissue.
Expression of the GPR451ike/GPR63 receptor in various human cell lines HUVECS (human umbilical vein endothelial cells), HCAEC (human coronary artery endothelial cells), MHVEC-L (human microvascular endothelial cells from lung), HPAEC (human coronary artery smooth muscle cells), HPASMC (human pulmonary artery smooth muscle cells), HAOSMC (human aortic smooth muscle cells) were investigated.
Transcripts of the GPR451ike/GPR63 receptor were detectable in all cell lines.
GPR451ike/GPR63 receptor expression in peripheral human tissue Expression of the receptor was determined using the TaqMan RT-PCR analysis semiquantitatively in relation to the expression in the brain.
An internal comparison took place in relation to the f3-actin RNA. Each measurement was determined twice. The following table shows the RNA expression as a multiple of the expression in the brain.
Table:
Expression of the GPR451ike/GPR63 receptor in various tissues in relation to the brain Expression relative to brain Brain 1 Heart 0.1 Kidney 0.2 Liver 0.01 Lung 0.05 Spleen 0.2 Thymus 0.6 Skeletal muscle 0.05 Pancreas 0.15 Small intestine 0.8 Stomach 0.7 .
Relatively strong expression was found in thymus, small intestine and stomach, while almost no expression was detectable in liver, kidney and skeletal muscle.
GPR451ike/GPR63 receptor expression in various parts of the brain.
Expression of the receptor was determined using the TaqMan RT-PCR
semiquantitatively in relation to expression in the cerebellum. The internal comparison was in relation to the f3-actin RNA. Each measurement was determined twice. The following table shows the RNA to the cerebellum.
Table:
Expression of the GPR451ike/GPR63 receptor in various parts of the brain in relation to the cerebellum Expression relative to the cerebellum Cerebellum 1 Whole brain 1.2 Corpus callosum 0.2 Caudatus 1.5 Thalamus 2.1 Amygdala 1.2 The strongest expression by comparison is observed in the thalamus.
Stimulation of the intracellular Ca2+ release by the GPR451ike/GPR63 receptor 209 different bioactive lipids from a substance library were added, each in a final concentration of 1 NM, to CHO cells which expressed the GPR451ikeIGPR63 receptor and the G Protein a subunit delta 6qi4mgr (= i49i4). About 30 compounds were tested. Only S1 P (sphingosine 1-phosphate) and DHS1 P
(dihydrosphingosine 1-phosphate) led to a measurable Ca2+ influx. S1 P and DHS1 P are naturally occurring lipid-like substances which also appear as natural ligands of other GPCRs.
The a subunit delta 6qi4mgr (= i4qi4) has a broad specificity in relation to different GPCRs.
Induction of cell growth by GPR451ike/GPR63 receptors.
An amount of 8 x 104 CHO cells was seeded onto 35 mm plates. The cells were transfected after 32 hours with 1 Ng of DNA of the vector construction. After hours, the cells were washed once with PBS and incubated in Iscove's medium containing 10% dialyzed fetal calf serum in the presence or absence of 1 NM S1 P for a further 48 hours. It was possible to show that test mixtures which contained contained about 20% more cells in the average of 2 tests done independently of one another.
SEQUENCE LISTING
<110> Aventis Pharma Deutschland GmbH
< 12 0 > Method for identif)ring agonists or antagonists of the GPR45 Iike/GP63 receptor <130> DEAV2002J0033 <140>
<141>
<160> 12 c170> PatentIn Ver. 2.1 <210> 1 c211> 1260 <212> DNA
<213> Homo sapiens <400> 1 atggtcttct cggcagtgtt gactgcgttc cataccggga catccaacac aacatttgtc 60 gtgtatgaaa acacctacat gaatattaca ctccctccac cattccagca tcctgacctc 120 agtccattgc ttagatatag ttttgaaacc atggctccca ctggtttgag ttccttgacc 180 gtgaatagta cagctgtgcc cacaacacca gcagcattta agagcctaaa cttgcctctt 240 cagatcaccc tttctgctat aatgatattc attctgtttg tgtcttttct tgggaacttg 300 gttgtttgcc tcatggttta ccaaaaagct gccatgaggt ctgcaattaa catcctcctt 360 gccagcctag cttttgcaga catgttgctt gcagtgctga acatgccctt tgccctggta 420 actattctta ctacccgatg gatttttggg aaattcttct gtagggtatc tgctatgttt 480 sttctggttat ttgtgataga aggagtagcc atcctgctca tcattagcat agataggttc 540 cttattatag tccagaggca ggataagcta aacccatata gagctaaggt tctgattgca 600 gtttcttggg caacttcctt ttgtgtagct tttcctttag ccgtaggaaa ccccgacctg 660 cagatacctt cccgagctcc ccagtgtgtg tttgggtaca caaccaatcc aggctaccag 720 gcttatgtga ttttgatttc_ tctcatttct ttcttcatac ccttcctggt aatactgtac 780 tcatttatgg gcatactcaa cacccttcgg cacaatgcct tgaggatcca tagctaccct 840 gaaggtatat gcctcagcca ggccagcaaa ctgggtctca tgagtctgca gagacctttc 900 cagatgagca ttgacatggg ctttaaaaca cgtgccttca ccactatttt gattctcttt 960 gctgtcttca ttgtctgctg ggccccattc accacttaca gccttgtggc aacattcagt 1020 aagcactttt actatcagca caactttttt gagattagca cctggctact gtggctctgc 1080 tacctcaagt ctgcattgaa tccgctgatc tactactgga ggattaagaa attccatgat 1140 gcttgcctgg acatgatgcc taagtccttc aagtttttgc cgcagctccc tggtcacaca 1200 aagcgacgga tacgtcctag tgctgtctat gtgtgtgggg aacatcggac ggtggtgtga 1260 <210> 2 <211> 4i9 <212> PRT
<213> Homo Sapiens <400> 2 Met Val Phe Ser Ala Val Leu Thr Ala Phe His Thr Gly Thr Ser Asn Thr Thr Phe Val Val Tyr Glu Asn Thr Tyr Met Asn Ile Thr Leu Pro Pro Pro Phe Gln His Pro Asp Leu Sez Pro Leu Leu Arg Tyr Ser Phe Glu Thr Met Ala Pro Thr Gly Leu Ser Ser Leu Thr Val Asn Ser Thr Ala Val Pro Thr Thr Pro Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu 65 70 T5 . 80 Gln Ile Thr Leu Ser Ala Ile Met Ile Phe Ile Leu Phe Val Ser Phe Leu Gly Asn Leu Val Val ors Leu Met Val Tyr Gln Lys Ala Ala Met Arg Ser Ala Ile Asn Ile Leu Leu Ala Ser Leu Ala Phe Ala Asp Met Leu Leu Ala Val Leu Asn Met Pro Phe Ala Leu Val Thr Ile Leu Thr Thr Arg Trp Ile Phe Gly Lys Phe Phe Cys Arg Val Ser Ala Met Phe Phe Trp Leu Phe Val Ile Glu Gly Val Ala Ile Leu Leu Ile Ile Ser Ile Asg Arg Phe Leu Ile Ile Val Gln Azg Gln Asp Lys Leu Asn Pro Tyr Arg Ala Lys Val Leu Ile Ala Val Ser Trp Ala Thr Ser Phe Cys Val Ala Phe Pro Leu Ala Val Gly Asn Pro Asp Leu Gln Ile Pro Ser Arg Ala Pro Gln Cys Val Phe Gly Tyr Thr Thr Asn Pro Gly Tyr Gln Ala Tyr Val Ile Leu Ile Ser Leu Ile Ser Phe Phe Ile Pro Phe Leu Val Ile Leu Tyr Ser Phe Met Gly Ile Leu Asn Thr Leu Arg His Asn 2fi0 2fi5 2?0 Ala Leu Arg Ile His Ser Tyr Pro Glu Gly Ile Cys Leu Ser Gln Ala Ser Lys Leu Gly Leu Met Ser Leu Gln Arg Pra Phe Gln Met Ser Ile 290 295 300 .
Asp Met Gly Phe Lys Thr Arg Ala Phe Thr Thr Ile Leu Ile Leu Phe Ala Val Phe Ile Val Cys Trp Ala Pro Phe Thr Thr Tyr Ser Leu Val Ala Thr Phe Ser Lys His Phe Tyr Tyr Gln His Asn Phe Phe Glu Ile Ser Thr Trp Leu Leu Trp Leu Cys Tyr Leu Lys Ser Ala Leu Aan Pro Leu Ile Tyr Tyr Trp Arg Ile Lys Lys Phe His Asp Ala Cys Leu Asp 370 375 380 .
Met Met Pro Lys Ser Phe Lys Phe Leu Pro Gln Leu Pro Gly His Thr Lys Arg Arg Ile Arg Pro 5er Ala Val Tyr Val Cys Gly Glu His Arg Thr Val Val <210> 3 <211> 42 <212> DNA
<213> Homo Sapiens <400> 3 ccgccgaagc ttgccatggt cttctcggca gtgttgactg cg 42 <210> 4 <211> 36 <212> DNA
<213> Homo Sapiens c4D0> 9 gccggcgaat tctcacacca ccgtccgatg ttcccc 36 <210>5 <211>24 <212>DNA
<213>Homo Sapiens <400> 5 cccactggtt tgagttcctt gacc 2g <210> 6 <211> 24 <212> DNA
<213> Homo Sapiens c400> 6 ggtagcctgg attggttgtg tacc 2q <210> 7 <211> 18 <212> DNA
<213> Homo Sapiens <400> 7 ctcaacaccc ttcggcac ig <210> 8 <211> 20 <212> DNA
<213> Homo Sapiens <400> 8 ggcctggctg aggcatatac 2D
<210> 9 <211> 28 <212> DNA
<213> Homo Sapiens <400> 9 tgccttgagg atccatagct accctgaa 2B
<210> 10 <211> 20 <212> DNA
<213> Homo sapiens <400> 10 tgcctggaca tgatgcctaa 2 <210> 11 <211> 19 <212> DNA
c213> Homo sapiens <400> 11 tccgtcgctt tgtgtgacc 19 c210> 12 c211> 25 <212> DNA
<213> Homo sapiens c400> 12 tccttcaagt ttttgccgca gctcc 25
Claims (18)
1. A method for identifying a compound which modifies the activity of the G
protein-coupled receptor GPR45like/GPR63, where a] biological material or a preparation of biological material which contains the G protein-coupled receptor GPR45like/GPR63 and a natural ligand of this receptor is provided, b] a chemical compound is provided;
c] the biological material or the preparation of biological material from a]
is brought into contact with the chemical compound from b];
d] the activity of the G protein-coupled receptor GPR45like/GPR63 is determined.
protein-coupled receptor GPR45like/GPR63, where a] biological material or a preparation of biological material which contains the G protein-coupled receptor GPR45like/GPR63 and a natural ligand of this receptor is provided, b] a chemical compound is provided;
c] the biological material or the preparation of biological material from a]
is brought into contact with the chemical compound from b];
d] the activity of the G protein-coupled receptor GPR45like/GPR63 is determined.
2. The method as claimed in claim 1, wherein the receptor is selected from one of the following groups a] a receptor consisting of the amino acid sequence as shown in SEQ ID
NO. 2, b] a receptor which is lacking one or more amino acids in relation to the amino acid sequence of SEQ ID NO. 2, c] a receptor in which one or more amino acids have been added in relation to the amino acid sequence of SEQ ID NO. 2, d] a receptor in which one or more amino acids have been replaced by other amino acids in relation to the amino acid sequence of SEQ ID
NO. 2.
NO. 2, b] a receptor which is lacking one or more amino acids in relation to the amino acid sequence of SEQ ID NO. 2, c] a receptor in which one or more amino acids have been added in relation to the amino acid sequence of SEQ ID NO. 2, d] a receptor in which one or more amino acids have been replaced by other amino acids in relation to the amino acid sequence of SEQ ID
NO. 2.
3. The method as claimed in claim 1, wherein the receptor is encoded by a polynucleotide sequence from one of the following groups a] a polynucleotide sequence as shown in SEQ ID NO. 1;
b] a polynucleotide sequence as shown in SEQ ID NO. 1 in which, owing to the degeneracy of the genetic code, one or more of the nucleotide positions contain a different nucleotide;
c] a polynucleotide sequence which hybridizes under stringent conditions with a polynucleotide sequence consisting of SEQ ID NO. 1.
b] a polynucleotide sequence as shown in SEQ ID NO. 1 in which, owing to the degeneracy of the genetic code, one or more of the nucleotide positions contain a different nucleotide;
c] a polynucleotide sequence which hybridizes under stringent conditions with a polynucleotide sequence consisting of SEQ ID NO. 1.
4. The method as claimed in one or more of claims 1 to 3, wherein the receptor is produced by prokaryotic or eukaryotic expression of an exogenous DNA
sequence comprising a polynucleotide sequence as shown in SEQ ID No. 1.
sequence comprising a polynucleotide sequence as shown in SEQ ID No. 1.
5. The method as claimed in claim 5, wherein the natural ligand is added from outside after biological material or a preparation of biological material which in each case comprises the G protein-coupled receptor GPR45like/GPR63 has been provided.
6. The method as claimed in one or more of claims 1 to 6, where the natural ligand is sphingosine 1-phosphate or lysophosphatidic acid.
7. The method as claimed in claim 6, where the natural ligand comprises a radioactive label or a label detectable by fluorimetry.
8. The method as claimed in one or more of claims 1 to 7, where the compound which modifies the activity of the G protein-coupled receptor GPR45like/GPR63 acts as agonist or antagonist of the natural ligand.
9. A compound which modifies the activity of the G protein-coupled receptor GPR45like/GPR63, where this compound has been identified by a method as claimed in claims 1 to 8.
10. The compounds as claimed in claim 9, whose mass is between 0.1 to 50 kDa.
11. The compound as claimed in claim 9 or 10, whose mass is between 0.1 to kDa.
12. The compound as claimed in claim 9, 10 or 11, whose mass is between 0.1 to 3 kDa.
13. The compound as claimed in one or more of claims 9 to 12, where this compound is 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.
14. A medicament comprising at least one compound as claimed in claims 9 to 13, together with excipients for formulating a medicament and/or further additives for the treatment of a cardiovascular disorder.
15. The use of a compound as claimed in claim 9 to 13 for producing a medicament for the treatment of a cardiovascular disorder.
16. The use of a compound which has been identified by a method as claimed in one of claims 1 to 5 for producing a complex of the receptor GPR45like/GPR63 with this compound.
17. The use of a compound as claimed in claim 16, wherein the compound is sphingosine 1-phosphate or lysophosphatidic acid.
18. A protein comprising a complex of the receptor GPR45like/GPR63 and sphingosine 1-phosphate and/or lysophosphatidic acid.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10225651A DE10225651A1 (en) | 2002-06-08 | 2002-06-08 | Method of identifying agonists or antagonists for the GPR45like / GPR63 receptor |
| DE10225651.9 | 2002-06-08 | ||
| PCT/EP2003/005600 WO2003104480A1 (en) | 2002-06-08 | 2003-05-28 | Method for identifying agonists or antagonists for the gpr45-like/gpr63 receptor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2488883A1 true CA2488883A1 (en) | 2003-12-18 |
Family
ID=29594349
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002488883A Withdrawn CA2488883A1 (en) | 2002-06-08 | 2003-05-28 | Method for identifying agonists or antagonists for the gpr45-like/gpr63 receptor |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP1516061B1 (en) |
| JP (1) | JP4472521B2 (en) |
| AT (1) | ATE403738T1 (en) |
| AU (1) | AU2003274087A1 (en) |
| CA (1) | CA2488883A1 (en) |
| DE (2) | DE10225651A1 (en) |
| DK (1) | DK1516061T3 (en) |
| ES (1) | ES2311102T3 (en) |
| WO (1) | WO2003104480A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106544359B (en) * | 2015-09-22 | 2019-07-19 | 复旦大学 | The purposes of GPR45 gene |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4138621A1 (en) * | 1991-11-25 | 1993-06-17 | Boehringer Ingelheim Int | METHOD FOR SCREENING SUBSTANCES WITH MODULATING EFFECT ON A RECEPTACLE-RELATED CELLULAR SIGNAL TRANSMISSION PATH |
| KR100629185B1 (en) * | 1997-11-11 | 2006-09-28 | 오노 야꾸힝 고교 가부시키가이샤 | Human lysophosphatidic acid receptor and use thereof |
| US6126587A (en) * | 1998-04-08 | 2000-10-03 | U.S. Centrifuge | Centrifugal separator apparatus including a plow blade assembly |
| AU4169499A (en) * | 1998-06-22 | 2000-01-10 | Japan Tobacco Inc. | Novel g protein-coupled receptor protein, dna and use thereof |
| EP1196572A1 (en) * | 1999-07-27 | 2002-04-17 | Smithkline Beecham Plc | Axor21, a g-protein coupled receptor |
| PT1263752E (en) * | 2000-03-17 | 2008-03-18 | Univ Tennessee Res Corp | Lpa receptor agonists and antagonists and methods of use |
| WO2002034781A1 (en) * | 2000-10-20 | 2002-05-02 | Solvay Pharmaceuticals Gmbh | Human g-protein coupled diadenosine tetraphosphate receptor (ap4ar) |
-
2002
- 2002-06-08 DE DE10225651A patent/DE10225651A1/en not_active Withdrawn
-
2003
- 2003-05-28 AT AT03740154T patent/ATE403738T1/en not_active IP Right Cessation
- 2003-05-28 EP EP03740154A patent/EP1516061B1/en not_active Expired - Lifetime
- 2003-05-28 DE DE50310285T patent/DE50310285D1/en not_active Expired - Lifetime
- 2003-05-28 WO PCT/EP2003/005600 patent/WO2003104480A1/en active IP Right Grant
- 2003-05-28 CA CA002488883A patent/CA2488883A1/en not_active Withdrawn
- 2003-05-28 JP JP2004511539A patent/JP4472521B2/en not_active Expired - Lifetime
- 2003-05-28 DK DK03740154T patent/DK1516061T3/en active
- 2003-05-28 AU AU2003274087A patent/AU2003274087A1/en not_active Abandoned
- 2003-05-28 ES ES03740154T patent/ES2311102T3/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| DE10225651A1 (en) | 2003-12-24 |
| JP2005528920A (en) | 2005-09-29 |
| DE50310285D1 (en) | 2008-09-18 |
| DK1516061T3 (en) | 2009-01-19 |
| AU2003274087A1 (en) | 2003-12-22 |
| EP1516061A1 (en) | 2005-03-23 |
| WO2003104480A1 (en) | 2003-12-18 |
| JP4472521B2 (en) | 2010-06-02 |
| ES2311102T3 (en) | 2009-02-01 |
| EP1516061B1 (en) | 2008-08-06 |
| ATE403738T1 (en) | 2008-08-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Geng et al. | Cloning and characterization of the human soluble adenylyl cyclase | |
| Daviet et al. | Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor | |
| Contos et al. | Lysophosphatidic acid receptors | |
| Vandorpe et al. | cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1: roles in regulatory volume decrease and erythroid differentiation | |
| Wu et al. | Functional significance of human trp1 and trp3 in store-operated Ca2+ entry in HEK-293 cells | |
| Schmidlin et al. | Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor | |
| SE523013C2 (en) | Nucleic Acid (IIP-10) Encoding an IGF-1 Receptor Binding Polypeptide and Using the Nucleic Acid or Polypeptide in a Method for Detecting the Proliferative Potential of a Cancer Cell or to Identify Substances That Modulate the Interaction between IIP-10 and IGF-1R | |
| US6485922B1 (en) | Methods for detecting compounds which modulate the activity of an LPA receptor | |
| US5912122A (en) | Nucleic acids encoding and method for detecting nucleic acid encoding human metabotropic glutamate receptor subtype mGluR6 | |
| WO1999019513A2 (en) | Methods for detecting compounds which modulate the activity of an lpa receptor | |
| JP2002500041A (en) | Identification of lysolipid receptor involved in inflammatory response | |
| Ruder et al. | EBAG9 adds a new layer of control on large dense-core vesicle exocytosis via interaction with Snapin | |
| US20030008350A1 (en) | 14273 receptor, a novel G-protein coupled receptor | |
| JP2001525178A (en) | Method for searching for agonist and antagonist for human 11cb splice variant | |
| US6448005B1 (en) | 14723 Receptor, a novel G-protein coupled receptor | |
| JPH11235184A (en) | Cdna clone hneaa81 encoding human 7-transmembrane receptor | |
| KR100790329B1 (en) | Use of EDG8 receptor | |
| US20020182655A1 (en) | Method for identifying ligands for G protein-coupled receptors | |
| CA2488883A1 (en) | Method for identifying agonists or antagonists for the gpr45-like/gpr63 receptor | |
| JPH1175870A (en) | New human g-protein-binding receptor (hcept09) | |
| Devignot et al. | A novel glycine receptor αZ1 subunit variant in the zebrafish brain | |
| US7300764B2 (en) | Method for identifying agonists and antagonists of the GPR45-like/GPR63 receptor | |
| Ding et al. | Isoform-specific regulation of adenylyl cyclase function by disruption of membrane trafficking | |
| JPH11127868A (en) | 7 tm receptor hlwar 77 | |
| US7794927B2 (en) | Method for identifying agonists or antagonists of the G-protein coupled receptor mas-like 1 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| AZWI | Withdrawn application |