EP1393077A2 - Verfahren zur identifizierung von liganden für g protein coupled receptors - Google Patents
Verfahren zur identifizierung von liganden für g protein coupled receptorsInfo
- Publication number
- EP1393077A2 EP1393077A2 EP02738024A EP02738024A EP1393077A2 EP 1393077 A2 EP1393077 A2 EP 1393077A2 EP 02738024 A EP02738024 A EP 02738024A EP 02738024 A EP02738024 A EP 02738024A EP 1393077 A2 EP1393077 A2 EP 1393077A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- ligand
- cell
- gpcr
- protein
- gpr3
- 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- Withdrawn
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Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
-
- 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
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- 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
-
- 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
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
Definitions
- the invention relates to a method for identifying ligands for orphan GPCRs (G protein coupled receptors) using FLIPR (Fluorometric Imaging Plate Reader).
- GPCR G-protein coupled receptors
- GPCRs are a family of integral membrane proteins located on the cell surface of cells. They receive signals from extracellular signal substances (e.g. hormones, neurotransmitters, peptides, lipids) and transmit these signals to the interior of the cell via a family of guanine nucleotide-binding proteins, so-called G-proteins. They activate different signal transduction pathways depending on the specificity of the receptor, the activated G protein and the cell type.
- the polypeptide chain of all GPCRs fold into seven ⁇ -helices that span the phospholipid double layer of the cell membrane. The seven membrane passages result in extra- and intracellular loops that enable extracellular ligand binding and the intracellular coupling of G proteins.
- GPCRs are also called seven-transmembrane receptors. All G protein-coupled receptors work according to a common basic pattern: the binding of an extracellular ligand leads to a change in the conformation of the receptor protein so that it can make contact with a G protein. A G-protein-mediated signal transduction cascade within the cell ultimately leads to a biological response from the cell.
- G-proteins are heterotrimeric proteins, which consist of the subunits, ß and ⁇ and are located on the inside of the cell membrane due to lipid anchors.
- the coupling of activated GPCRs to G proteins causes a GDP / GTP exchange of the G ⁇ subunit and the dissociation of the heterotrimer into an ⁇ and a ⁇ subunit. Both the activated ⁇ subunit and the ⁇ complex can influence intracellular effector proteins.
- G proteins of the gas type leads, for example, to an increase in the intracellular cAMP level or to a decrease in the latter when G proteins of the G ⁇ i type are activated.
- Gq-type G proteins activate phospholipase C (PLC), which catalyzes the formation of inositol-1, 4,5-triphosphate (IP3) and diacylglycerol (DAG). These molecules in turn lead to
- G protein kinase C PLC
- Promiscuity refers to the non-selectivity of the G protein towards a GPCR.
- promiscuous G proteins can be produced from hybrid G proteins or by mutagenesis within the G16 family. So z. B. by the fusion of the receptor recognition region of G ⁇ i with the effector activation region of G ⁇ q a G ⁇ q / i hybrid can be produced, which receives signals from Gi-coupled receptors but receives the G ⁇ q-PLCß signal transduction pathway.
- Such a hybrid, in which the C-terminal 5 amino acids of G ⁇ q has been replaced by the corresponding G ⁇ i sequence (G ⁇ qi5) was described for the first time by Conklin et al., Nature 363, 274-276 (1993).
- Adenylate cyclase is more easily accessible by measurement methods and can be used in high-throughput screening.
- One device that can measure intracellular Ca 2+ levels in the 96-well or 384-well formal is the FLIPR (Molecular Devices).
- GPCRs No natural ligands are known for many GPCRs. Such GPCRs are called orphan (English: Weisenkind) GPCRs.
- GPR stands for another name of G-protein-coupled-receptor. The numbers refer to the specific type. For all three receptors, a strong form was originally found in the central nervous system. In the meantime, however, it has been demonstrated that GPR 3, 6 and 12 are also expressed in the peripheral vascular system (endothelial cells and smooth muscle cells). It can therefore be assumed that these receptors play an essential role in the physiology / pathophysiology of the endothelium and thus of the entire vascular system of humans. The development of high blood pressure, atherosclerosis or other cardiovascular diseases could be associated with these receptors. Sequence comparisons revealed a high degree of homology to GPCRs with lipid ligands.
- GPR 3, 6 and 12 could also be lipid receptors.
- the sequence information for the genes of the three receptors is available to the public.
- the sequence number for the human gene of GPR 3 is the number “L 32831”, for the human gene GPR 6 the number “L 36150” and for the human gene of GPR 12 the number “U 18548”.
- the information is available for example via www.ncbi.nlm.nih.gov.
- ligands for GPCRs are usually determined in laboratory experiments by trial and error.
- the disadvantage of this approach is that it is often time-consuming and controlled by chance. It is therefore an object of the present invention to provide a quick and simple method by means of which one or more ligands for a GPCR can be identified.
- the incorporation of results from bioinformatics is indispensable for the assay setup and the targeted search for physiological ligands.
- Receptors should be able to be switched on by adding the activator exogenously.
- attempts were made to switch the receptors on the HTS-capable Ca 2+ pathway after stimulation with potential lipid activators.
- the functional experiments were carried out in the presence of 300 ⁇ M suramin in order to reduce the background present by endogenous lipid receptor expression and to potentiate the signals mediated by GPR3, 6, 12.
- a physiological ligand is also unknown for the rat GPR3.
- the invention therefore relates to a method for identifying a ligand of a G protein-coupled receptor (GPCRs)
- a cell is made available whose endogenous signal transduction cascades allow the coupling of the orphan GPCRs GPR3, 6 and 12 as well as rGPR3 to the Ca 2+ pathway. This is essential here
- the cell line from a) is transfected with a recombinant construct of a GPCR in such a way that said GPCR is overexpressed in this cell line, c) at least one chemical compound is made available which, based on theoretical considerations, is supported by bioinformatics and literature research (Eggerickx et al, 1995) comes into consideration as a possible ligand.
- Fluorescence measurement from e) is carried out with the results of a fluorescence measurement after contacting a cell according to a) with a chemical compound according to c).
- the GPCRs of the present invention interact particularly with the G ⁇ i class of the G protein ⁇ subunits.
- the activation of G ⁇ i causes the inhibition of the adenylate cyclase.
- the ß ⁇ complex can in turn cause an intracellular release of Ca2 + from storage organelles.
- G alpha proteins are used, the GPCRs of different ligand specificity at the
- the protein "G ⁇ 16" is particularly suitable for carrying out a method of the aforementioned invention. Further G ⁇ proteins that can be used to carry out the method are disclosed in DE 10033353.2.
- a GPCR for which no physiological ligand is known is preferably used for carrying out the method.
- a physiological ligand is to be understood as a molecule which is formed by an organism, in particular a mammal, which binds to this GPCR and thereby activates a downstream G alpha protein.
- a GPCR from the group GPR 3, 6 or 12 is furthermore preferably suitable for implementation.
- the complete receptor of the GPR 3 from rat is particularly preferred.
- Those of mammals are preferably suitable as cells. These cells can consist of primary cells or cell lines. Examples of such cells are primary cells from mammalian organs (e.g. brain, muscle, adipose tissue, heart, lungs, liver, Kidney, blood vessels, hormone glands, etc.). Suitable cell lines are preferably CHO, HEK 293, COS, mouse 3T3, Heia cells or others. Yeast cells can furthermore preferably be used.
- the provision of a cell comprises its manufacture, cultivation and further processing. It is provided, for example, by preparing suitable cell material from organs or tissues or by multiplying suitable cell lines or microorganisms. Various suitable nutrient media can be used for cultivation. The cells are kept at the optimal temperature for the organism. If necessary, preservatives, antibiotics, pH indicators, blood serum components, blood serum, auxiliaries or others are added to the growth medium used in each case. Methods for the production, cultivation and further processing are described in standard works. (Example: Basic Cell Culture; Ed. J.M. Davis; IRL Press; 1994).
- a construct to be expressed in a cell is in the form of a polynucleotide sequence that can be routinely produced by the person skilled in the art with the help of his specialist knowledge.
- the specialist knowledge of the biochemical / molecular biological specialist for this can be found, for example, in “FM Ausubel et al.; Current Protocols in Molecular Biology; John Wiley &Sons; New York ".
- a polynucleotide which codes for the amino acid sequence of a GPCR for example, is incorporated into an expression vector.
- An expression vector is to be understood as a vector in which a polynucleotide sequence can be transfected and expressed in a host cell.
- Vectors can be derived of plasmids, viruses, or cosmids. Vectors must have the ability for autonomous replication. They generally contain an origin of replication, interfaces for restriction enzymes and marker genes such as genes for antibiotic resistance Functional control of a promoter
- a promoter is a functional polynucleotide sequence of different lengths by means of which the transcription, ie the synthesis of mRNA, is directly in 3 ' direction behind the promoter polynucleotide sequence is controlled. There are promoters that are only active in procarotes, such as the lac, tac or trc promoter, and promoters that are only active in eukaryotes, such as the CMV, T, or ADH promoter.
- 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 an organism, for example a bacterium, fungus or the cells of a eukaryotic cell line.
- the promoter can be inducible, for example by means of tryptophan, or it can be constitutively active.
- expression vectors are pUC18, pUC19, pBluesccript, pcDNA3.1 or others.
- Transfection is understood to be the introduction of foreign polynucleotide sequences into a host cell by means of a vector and the subsequent multiplication of this polynucleotide sequence to any number of identical copies.
- transient transfection of a cell line with a recombinant construct takes place by means of routine methods which the person skilled in the art has mentioned in the above-mentioned "Current Protocols in Molecular Bioiogy” published by John Wiley & Sons, New York or in “Sambrook et al., A Laboratory Mannual, Cold Spring Harbor Laboratory , ISBN 0- 87969-309-6 "finds.
- routine procedures are, for example, electroporation, Ca2 + phosphate coprecipitation or transfection using liposomes.
- Expression of transfected genes in the host cell can be detected by Western blotting of cell lysates from transfected cells in combination with an immunological detection method.
- a chemical compound is made available in particular by chemical synthesis or by isolation of chemical substances from biological material.
- Biological material contains living or non-living cells or components thereof.
- Suitable solvents can be water, buffer substances (e.g. Tris, Hepes, Mops etc.) monovalent and / or divalent ions (e.g. K + , Na + , Mg 2+ , Ca 2+ etc.), acids (e.g. HCL, H2SO4,
- alkalis e.g. NaOH etc.
- alcohol e.g. methanol, ethanol, glycerin
- detergents e.g. Na-dodecyl sulfate etc.
- organic solvents e.g. formamide, acetone, Dimethyl sulfoxide, etc.
- other components in particular for solubilization or stabilization.
- a chemical compound for the process according to the invention should be suitable as a ligand for a GPCR. This chemical compound could also be a lipid due to the similarity of GPR
- Such a connection can in particular from tissues or organs of vertebrates such.
- B. endothelial cell tissue, heart tissue, brain tissue, blood, serum or plasma can be obtained.
- the natural ligand of a GPCR is preferably suitable for carrying out the method according to the invention.
- the person skilled in the art can use routine methods from the laboratory to bring the chemical compound into contact with the cell line mentioned. The contacting takes place, for example, in Erlenmayer vessels, tubes, Eppendorf vessels or on microtiter plates. Temperature-controlled incubators can be used to bring them into contact, in which a constant temperature of, for example, 30 ° C. or 37 ° C. and uniform CO 2 conditions or humidity conditions can be regulated.
- the contacting can in particular also be carried out in devices provided for this purpose in a laboratory robot (FLIPR).
- FLIPR laboratory robot
- the contacting is possible over different time spans of a few seconds, over minutes up to several hours.
- the conditions to be selected depend on the receptor, the cell line and the chemical compound.
- FLIPR Fluometric Imaging Plate Reader
- the system is suitable for measuring intracellular Ca 2+ signals. The determinations are carried out in microtiter plates with 96 or 384 wells.
- the binding of a ligand to a GPCR leads to the intracellular release of Ca 2+ .
- the amount of Ca + released can be determined using a calcium-sensitive fluorescent dye (e.g. fluo- 4).
- GPCR can be determined.
- a ligand activates the Ca 2+ release much more strongly in the cell, which functionally overexpresses a GPCR.
- the technical equipment of the FLIPR system including the reagents for the determination of Ca 2+ are commercially available.
- One provider is in particular the company Molecular Devices with offices in Sunnyvale (CA), Ismaning (AE) and Ashiya (JP), among others.
- the invention also relates to a ligand identified by the method described above.
- S1 P and DHS1 P cause Ca 2+ release in HEK293 cells.
- Suramin was used in the present assay to reduce the endogenous lipid background.
- a natural ligand is used by an organism e.g. B. a vertebrate to appropriately activate a GPCR in the context of the organism.
- the purpose is there especially in the initiation of biochemical functions such.
- B. the activation of action potentials to process sensory stimuli, the activation of a gene synthesis for a structural or messenger protein, the release of messenger substances, the regulation of metabolic functions, the regulation of organ functions such as the heartbeat of blood pressure or similar biological processes.
- a chemical compound that is suitable as a ligand for a GPCR binds to this GPCR. Binding of a ligand triggers activation of this receptor.
- the activation of a GPCR leads to the release of intracellular Ca2 + in the cell in the method according to the invention.
- the invention further relates to a medicament which contains a ligand mentioned above and furthermore additives for stabilizing the ligand and / or for formulating a medicament.
- the invention also relates to the manufacture of a medicament. For this purpose, the above-mentioned ligand is mixed with the additives, then the drug is processed to its final form, filled, provided with an instruction leaflet and packaged.
- the final form of a drug relates to the final formulation, for example as a tablet, granulate, spray, solution, ointment, tincture or other formulation forms.
- the processing to the final form refers to the production of the respective formulation.
- the invention also includes the use of a ligand as mentioned above for the manufacture of a medicament which is suitable for the treatment of a disease which is based on a malfunction of the GPCR to which this ligand binds.
- the ligand is preferably used for the production of a medicament for the treatment of cardiovascular diseases and CNS diseases.
- the ligand is preferably in the form of a pharmaceutical composition with an acceptable carrier.
- the carrier must of course be compatible, in the sense that it is compatible with the other components of the composition and is not harmful to the health of the patient.
- the carrier can be a solid or a liquid or both and is preferably formulated with the compound as a single dose, for example as a tablet, ranging from 0.05% to Can contain 95% by weight of the active ingredient.
- compositions according to the invention can be prepared by one of the known pharmaceutical methods, which essentially consist in mixing the constituents with pharmacologically acceptable carriers and / or auxiliaries.
- Pharmaceutical compositions according to the invention are those which are suitable for oral, rectal, topical, peroral (e.g. sublingual) and parenteral (e.g. subcutaneous, intramuscular, intradermal or intravenous) administrations.
- the amount of a ligand, as mentioned above, required to achieve a desired biological effect depends on a number of factors such as e.g. The specific compound chosen, the intended use, the mode of administration or the clinical condition of the patient.
- the quantities given below relate to a ligand.
- the daily dose is in the range from 0.3 mg to 100 mg (typically from 3 mg to 50 mg) per day per kilogram of body weight, e.g. B. 3-10 mg / kg / day.
- An intravenous dose can e.g. B. are in the range of 0.3 mg to 1.0 mg / kg, which can suitably be administered as an infusion of 10 ng to 100 ng per kilogram per minute.
- Suitable infusion solutions for these purposes can e.g. B. from 0.1 ng to 10 mg, typically from 1 ng to 10 mg per milliliter.
- Single doses can e.g. B. from 1 mg to 10 g of the active ingredient.
- ampoules for injections may contain, for example, from 1 mg to 100 mg, and orally administrable egg dose formulations, such as tablets or capsules, for example, from 1.0 to 1000 mg, typically from 10 to 600 mg.
- the invention also relates to a polynucleotide sequence according to Seq ID No. 1, which codes for a GPR 3 of the rat.
- the invention further comprises a protein for the GPR 3 of the rat containing at least one amino acid sequence according to Seq ID No. 2.
- the invention also contains an expression vector in which the above-mentioned polynuclide sequence of the GPR 3 of the rat according to Seq ID No. 1 was incorporated.
- the invention also relates to the production of a cell which expresses the GPR 3 of the rat containing at least the amino acid sequence according to Seq ID No. 2, this cell being transfected by means of the expression vector mentioned above.
- the human genes of GPR 3, 6 and 12 do not contain introns. They can therefore be amplified from human genomic DNA using PCR (polymerase chain reaction). The coding sequences were cloned via the Hind III / Xbal sites of pcDNA 3.1 (Invitrogen).
- the sequence of the complete gene of GPR 3 in the rat was determined by PCR amplification of cDNA of the rat brain using the ⁇ 'primer 5' -ATG GCC CTT ATG GCC TGG TTC GCC TCA GCC TCA-3 '(Seq ID3) and the 3 ' Primers (mouse) 5 ' -TCT AGA CTA GAC ATC ACT AGG GGA CCG GGA-3 '(Seq ID4) increased.
- the ⁇ ' primer additionally provides a Hind III site and generates a Kozac sequence before the start codon. An Xbal position is also taken into account in the 3 ' primer.
- the 990 bp long amplification product was cloned into the Hind III / Xbal sites of pcDNA 3.1 (Invitrogen) and then sequenced.
- HEK293 cells (cell line from Human Embryonic Kidneys) were cultivated in DMEM, which was supplemented with 10% fetal bovine serum 10,000 lU / ml penicillin, 10,000 ⁇ g / ml streptomycin and 25 mM Hepes pH 7.0.
- CHO-K1 cells (cell line from Chinese Hamster Ovary) were cultivated in Iscove medium, which was supplemented with 10% fetal bovine serum, 10,000 lU / ml penicillin, 10,000 ⁇ g / ml streptomycin, gentamycin and 2 mM L-glutamine.
- Iscove medium is commercially available e.g. B. of biochrom.
- the transfection was in the case of HEK293 cells using FuGene 6 transfection reagent (commercially available, for example from Röche Diagnostics) and in the case of CHO cells using Lipofectamine reagent (commercially available, for example from GIBCO-BRL) according to the instructions in the Manufacturer carried out.
- FLIPR Fluorometric Imaging Plate Reader
- HEK293 cells were transferred to 96 well microtiter plates 24 hours after transient transfection.
- the cell density was 80,000 cells per well.
- Microtiter plates were coated with poly-D-lysine.
- the cells were kept in medium with 1% FCS (fetal calf serum) for 18 to 24 hours. After this The cells were trypsinized in DMEM (Dulbecco 's modified Eagle medium), which additionally contains 25 mM HEPES (N-2-hydroxyethylpiperazine-N ' -2-ethanesulfonic acid) pH 7.0, 1% FCS, 10,000 lU / ml of penicillin, 10,000 ⁇ g / ml streptomycin and 4 ⁇ M dye fluo4 (fluorescent calcium indicator from Molecular Dynamics) contained suspended and then incubated for 1 hour at 37 ° C. under 5% CO 2.
- DMEM Dulbecco 's modified Eagle medium
- HEPES N-2-hydroxyethylpiperazine-N ' -2-ethanesulfonic acid
- PBS phosphate buffered saline
- FAF-BSA bovine serum albumin free
- Fatty acids contained. After the last washing step, the volume was 100 ⁇ l per well.
- the compounds to be examined were mostly in the form of 2 mM stock solutions in DMSO (dimethyl sulfoxide). This was diluted 1: 500 with the PBS solution, which contained 1 mM MgCl2 1 mM EDTA and 0.4 mg / ml FAF-BSA. Lipids were pre-assayed as triple-concentrated solutions.
- Suramin (Sigma) was prepared in PBS containing 1 mM MgCl2, 1 mM EDTA and 0.4 mg / ml FAF BSA as a triple-concentrated solution.
- the FLIPR was programmed in such a way that 50 ⁇ l from the triple concentrated stock solution of suramin was added to the cells. This gave a final concentration of 300 ⁇ M with respect to suramin. Fluorescence was measured at 3 second intervals for the first 3 minutes and at 10 second intervals for the last 2 minutes. Ca2 + signals from ligands were determined analogously. The fluorescence measurements of the time interval from 18 seconds to 37 seconds were used to determine agonist activity. The device itself carries out an internal comparison of the zero values.
- GPCR 3, 6 and 12 are orphan receptors of the GPCR protein family With the help of Genbank searches (EMBL), the lipid receptors EDG, (endothelial differentiation gene) and cannabinoid GPCRs were found to be the structurally closest relatives of GPR 3, 6 and 12 be identified. Within one Species homology is 65-68% based on the nucleotide sequence. In comparison to the rat sequences, the human sequences each show 87% (GPR 3), 83% (GPR 6) and 88% (GPR 12) homology.
- RT-PCR Reverse transcripase polymerase chain reaction
- lipid library consisting of 200 bioactive lipids did not provide any further ligands and also showed the ineffectiveness of cannabinoids.
- the newly cloned rGPR 3 (L 32829, 33 bp partial sequence), like its human homolog, can be switched on by S1 P / DHS1 P in a Ca 2+ FLIPR assay.
- the coupling of GPR 3, 6 and 12 to the Ca 2+ signaling pathway works in HEK293 cells without the co-transfection of G-Q or other chimeras, promiscous G ⁇ subunits.
- the S1 P / DHS1 P-induced Ca 2+ release in HEK293 cells is sensitive to pertussis toxin, ie the signal transduction cascade runs via G proteins of the G ⁇ i type.
- the Ca 2+ release is partially sensitive to inhibitors of sphingosine kinase.
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Abstract
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Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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DE10123958A DE10123958A1 (de) | 2001-05-17 | 2001-05-17 | Verfahren zur Identifizierung von Liganden für G protein coupled receptors |
DE10123958 | 2001-05-17 | ||
PCT/EP2002/004845 WO2002092831A2 (de) | 2001-05-17 | 2002-05-03 | Verfahren zur identifizierung von liganden für g protein coupled receptors |
Publications (1)
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EP1393077A2 true EP1393077A2 (de) | 2004-03-03 |
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EP02738024A Withdrawn EP1393077A2 (de) | 2001-05-17 | 2002-05-03 | Verfahren zur identifizierung von liganden für g protein coupled receptors |
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US (1) | US20020182655A1 (de) |
EP (1) | EP1393077A2 (de) |
JP (1) | JP2004535180A (de) |
AR (1) | AR035901A1 (de) |
AU (1) | AU2002312871A1 (de) |
DE (1) | DE10123958A1 (de) |
WO (1) | WO2002092831A2 (de) |
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DE10256947A1 (de) * | 2002-12-05 | 2004-06-24 | Grünenthal GmbH | Verfahren zur Messung der Aktivität G(alpha)i- oder G(alpha)o-gekoppelter Rezeptoren mittels Ca2+-Influx in Zellen |
US8580517B2 (en) * | 2003-02-05 | 2013-11-12 | Washington University In St. Louis | Biosensor and use thereof to identify therapeutic drug molecules and molecules binding orphan receptors |
EP1654225A4 (de) * | 2003-08-13 | 2007-11-28 | Amgen Inc | Antagonisten des melanin concentrating hormone-rezeptors |
CA2533112A1 (en) * | 2003-08-13 | 2005-03-03 | Christopher Hulme | Melanin concentrating hormone receptor antagonist |
US7063034B2 (en) * | 2004-08-10 | 2006-06-20 | Watermark Paddlesports, Inc. | Spray skirt for watercraft |
GB0421285D0 (en) * | 2004-09-24 | 2004-10-27 | Univ Nottingham | Improvements in high content screening |
GB0421693D0 (en) | 2004-09-30 | 2004-11-03 | Amersham Biosciences Uk Ltd | Method for measuring binding of a test compound to a G-protein coupled receptor |
US8221990B2 (en) | 2007-04-06 | 2012-07-17 | Teijin Pharma Limited | Screening GPR12 receptor for substances having Nesfatin-1-like action, or which regulate Nesfatin-1 action |
EP1986008A1 (de) * | 2007-04-06 | 2008-10-29 | Teijin Pharma Limited | Screening-Verfahren zur Identifizierung eines Nesfatin-1 Funktionsreglers oder einer Nesfatin-1 ähnlichen Substanz mittels eines Rezeptorproteins ausgewählt aus GPR3, GPR6 und GPR12 |
CN104732117B (zh) * | 2015-04-02 | 2018-02-13 | 苏州科技学院 | 基于反馈式条件随机场的g蛋白偶联受体拓扑计算预测方法 |
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JPH11503012A (ja) * | 1995-03-30 | 1999-03-23 | ヒューマン ジノーム サイエンシーズ,インコーポレイテッド | ヒトgタンパク質結合レセプター |
DE19610984A1 (de) * | 1996-03-21 | 1997-09-25 | Boehringer Mannheim Gmbh | Alkohol-Dehydrogenase und deren Verwendung zur enzymatischen Herstellung chiraler Hydroxyverbindungen |
US6121320A (en) * | 1997-02-26 | 2000-09-19 | The University Of Kentucky Research Foundation | Combination anti-leukemic therapy by utilizing suramin and biologic response modifiers |
ES2147167T1 (es) * | 1997-04-14 | 2000-09-01 | Dominic P Behan | Procedimiento de identificacion de moduladores de receptores de membrana de superficie celular util para el tratamiento de enfermedades. |
-
2001
- 2001-05-17 DE DE10123958A patent/DE10123958A1/de not_active Withdrawn
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2002
- 2002-05-03 EP EP02738024A patent/EP1393077A2/de not_active Withdrawn
- 2002-05-03 JP JP2002589697A patent/JP2004535180A/ja active Pending
- 2002-05-03 WO PCT/EP2002/004845 patent/WO2002092831A2/de not_active Application Discontinuation
- 2002-05-03 AU AU2002312871A patent/AU2002312871A1/en not_active Abandoned
- 2002-05-15 AR ARP020101785A patent/AR035901A1/es unknown
- 2002-05-16 US US10/146,065 patent/US20020182655A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
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See references of WO02092831A2 * |
Also Published As
Publication number | Publication date |
---|---|
WO2002092831A3 (de) | 2003-11-13 |
DE10123958A1 (de) | 2002-11-28 |
JP2004535180A (ja) | 2004-11-25 |
WO2002092831A2 (de) | 2002-11-21 |
US20020182655A1 (en) | 2002-12-05 |
AR035901A1 (es) | 2004-07-21 |
AU2002312871A1 (en) | 2002-11-25 |
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