EP1664333A2 - Criblage a haut rendement et sensibilite elevee pour mesurer l'activite kinase du recepteur beta-adrenergique et pour determiner l'impact sur cette activite de substances a tester - Google Patents

Criblage a haut rendement et sensibilite elevee pour mesurer l'activite kinase du recepteur beta-adrenergique et pour determiner l'impact sur cette activite de substances a tester

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Publication number
EP1664333A2
EP1664333A2 EP04764711A EP04764711A EP1664333A2 EP 1664333 A2 EP1664333 A2 EP 1664333A2 EP 04764711 A EP04764711 A EP 04764711A EP 04764711 A EP04764711 A EP 04764711A EP 1664333 A2 EP1664333 A2 EP 1664333A2
Authority
EP
European Patent Office
Prior art keywords
protein kinase
protein
receptor
antibody
substrate molecule
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Withdrawn
Application number
EP04764711A
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German (de)
English (en)
Inventor
Aimo Kannt
Antje Pommereau
Harald Thuering
Jochen Huber
Martin Oppermann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi Aventis Deutschland GmbH
Original Assignee
Sanofi Aventis Deutschland GmbH
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Publication date
Application filed by Sanofi Aventis Deutschland GmbH filed Critical Sanofi Aventis Deutschland GmbH
Priority to EP04764711A priority Critical patent/EP1664333A2/fr
Publication of EP1664333A2 publication Critical patent/EP1664333A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • High-Throughput-Assay with high sensitivity for measuring of the activity of ⁇ - adrenergic receptor kinase and for determining the impact of test substances on such activity.
  • the invention refers to a GPCR specific protein kinase assay by means of head linked substrate molecules.
  • GPCRs G-protein-coupled receptors
  • ⁇ -AR ?-adrenergic receptors
  • Decreased cardiac activity is mediated by the cholinergic parasympathetic system working through muscarinic acetylcholine GPCRs.
  • Cardiac ⁇ -AR consists of both the ⁇ -i and ⁇ subtypes, and in the human heart, as in most mammals, the ⁇ i is predominant.
  • the two proteins are rather similar in protein sequence, though the ⁇ -i is significantly larger due to a larger N-terminal extracellular region and a larger C3 cytoplasmic loop.
  • Trimeric G-proteins consist of an a, ⁇ and y subunit, each represented by several different genes in humans.
  • GRK G Protein Specific Receptor Kinase
  • GRK1 rhodopsin kinase
  • GRK2 ⁇ -adrenergic receptor kinase-1
  • GRK3 ⁇ - adrenergic receptor kinase-2
  • GRK4 GRK5; and GRK6.
  • GPRK1 and wj283.2 encode, respectively, Drosophila and Caenorhabditid elegans proteins with 62% and 51% homology to bovine GRK2. Similary, GPRK2 and f19c6.1 encode Drosophila and C. elegans proteins with 50-53% homology to bovine GRK5.
  • the amino acid sequence similarity is 53-93%, with GRK1 and GRK2 being the most divergent.
  • GRKs are most closely related to the PKC and cAMP-dependent protein kinase families (PKAs). Based on sequence and functional similarities, the GRK family has been divided into three subfamilies: (a] GRK1 , (b] GRK2 or ⁇ -adrenergic receptor kinase ( ⁇ -ARK); (GRK2 and GRK3), and (c] GRK4 (GRK4, GRK5, and GRK6). Members of the GRK2 subfamily share approximately 84% sequence similarity, and members of the GRK4 subfamily share approximately 70% sequence similarity.
  • PKAs PKC and cAMP-dependent protein kinase families
  • cardiac ⁇ -adrenergic receptors are stimulated by an increased release of catecholamines from the sympathetic nerves to maintain cardiac output and blood pressure.
  • This increase in ⁇ -adrenergic receptor agonists leads to a downregulation of the receptor (and thus to a lower receptor density) and a receptor desensitization mediated by overexpression of ⁇ ARK.
  • treatment with ⁇ ARK inhibitors may be of benefit for the preservation of myocardial ⁇ -adrenergic receptor signaling and may delay the development of heart failure after myocardial infarction.
  • GPT-binding-protein-coupled receptor kinase 2 (GRC2) binds and phosphorylates tubulin, Eur. J. Biochem. 255, 363-368" an assay for GRK2 has been described by means of radioactively labeled substrate molecules.
  • the AlphaScreen technology is widely used for testing of different types of enzymes.
  • AlphaScreen Assays of different specificity are commercially available e.g. from
  • AlphaScreen is a bead based non-radioactive Amplified Luminescent Proximity
  • a photosensitizer in the Donor bead converts ambient oxygen to a more excited singlet state.
  • the singlet state oxygen molecules diffuse across to react with a thioxene derivative in the Acceptor bead, generating chemiluminescence at 370 nm that further activates fluorophores contained in the same bead.
  • the fluorophores subsequently emit light at 520-620 nm.
  • AlphaScreen provides a highly versatile, sensitive, time-resolved, homogeneous and miniaturizable means to efficiently perform assay development and HTS resulting in higher throughput at lower costs.
  • the lifetime of singlet oxygen in solution is about 4 ⁇ s which corresponds to a diffusion distance of approximately 200nm. Thus, a signal is only generated when donor and acceptor beads are in close proximity to each other.
  • biotinylated CCR5 peptide (Seq. ID No. 1 ) is phosphorylated by the kinase.
  • the phosphorylated serine is recognized by a specific antibody which, in turn, is bound to protein A coupled acceptor beads.
  • the biotinylated N-terminus of the CCR5 peptide is coupled to streptavidin-coated donor heads.
  • the assay of the invention in particular the test of ⁇ ARK activity by means of the AlphaScreen technology design is new. Compared to the state of the art determination of the ⁇ ARK activity is much more sensitive since the change of substrate phosphorylation is detectable within the nM-range. Furthermore the assay is homogenous which means that additional washing steps or immobilization of reactants are easily avoidable since the reaction is performed by means of solid phases. The assay is suitable for miniaturiarisation which allows the use in high throughput screening by laboratory robots. A further advantage is that the substrate does not need to be modified by covalent binding to fluorescence dyes.
  • Method for detecting the phosphorylation of a substrate molecule by a protein kinase wherein a] at least one protein kinase is provided, b] at least one substrate molecule of the protein kinase of a] is provided, c] at least one donor component which is able to bind to the substrate molecule from b] and which emits a signal after the donor component was irradiated by a light source, d] at least one acceptor component which is able to recognize the substrate of b] in case it had been phosphorylated and which acceptor component is able to receive a signal in the form of an electromagnetic radiation is provided, e] the protein kinase from a], the substrate molecule from b], the donor component from c] and the acceptor component from d] are brought in contact, f] the mixture of e] is irradiated with a light source in such a way that the donor component of c] is excited, g] the electromagnetic radiation which is e
  • a substrate molecule shall mean any chemical compound which is phosphorylatable by a protein kinase.
  • a substrate molecule consists preferably of a peptide or a protein.
  • a substrate molecule may consist of a G-Protein coupled Receptor or a member of a signaling cascade.
  • a donor component is able by means of a donor compound to emit a signal after irradiation by a light source.
  • the signal may consist of an electromagnetic radiation which is characterized by a specific wavelength or may consist of an atom or of a molecule or another type of output.
  • An acceptor component is able to emit electromagnetic radiation upon having received the signal from the donor component.
  • This electromagnetic radiation is characterized by its specific wavelength. It can be detected by means of an analysis apparatus as e.g. by a spectrometer.
  • the substrate molecule can be linked to an antibody which is able to bind to the donor component or the donor component is linked to an antibody which is able to bind to the substrate molecule.
  • the substrate molecule can be linked to a first antibody and the donor component can be linked to a second antibody wherein either the first antibody is able to bind to the second antibody or the second antibody is able to bind to the first antibody.
  • the linking of a compound to one or several other compounds shall mean that the connection between these two compounds occurs permanently.
  • the linking is realized e.g. by covalent chemical bondages, or electrostatic forces as mediated by ionized molecules or by other means.
  • binding of a compound to one or several other compounds shall start from a non- binding stage, then continue to the contracting of the two compounds after finally forming a strong bondage between the said compounds.
  • This bondage may be characterized by binding constants which are preferably within range of 5x10 "9 to 5 «10 " 7 M.
  • the substrate component is linked to a biotin molecule and the donor component is linked to streptavidin or the substrate component is linked to streptavidin and the donor compound is linked to biotin.
  • Biotin and streptavidin can bind to each other.
  • Other chemical groups linked to the substrate component or donor component which are able to bind to each other comprise e.g. DNA and RNA binding proteins and the corresponding polynucleotide binding motifs.
  • the Protein kinase is e.g. the beta-Adrenerge-Receptor-Kinasel or the G-Protein- Coupled-Receptor-Kinase2.
  • the substrate can consist of a G-protein coupled Receptor or of the muscarinic Acetylcholine-Receptor or an adrenergic alpha 2 Receptor.
  • the acceptor component is linked to at least one antibody which specifically binds to the substrate of b] in case this substrate had been phosphorylated.
  • an antibody is e.g. the monoclonal antibody E11/19. Manufacturing of this antibody was described in "Pollak-Kapp. et al., J. Biol. Chem. (2003), 278, 2190-2198".
  • the electromagnetic radiation which is emitted by the mixture according to g] of the method of the invention for detecting the phosphorylation of a substrate molecule can be fluorescence radiation.
  • the electromagnetic radiation according to g] may be detected, for example, with the aid of a photomultiplier.
  • the light source for irradiation of the mixture according to f] of the method of the invention for detecting the phosphorylation of a substrate molecule can be a laser or a lamp.
  • the signal from the donor component to the acceptor component can be transferred preferably by emissionless energy transfer.
  • This energy transfer can be a fluorescence resonance energy transfer.
  • the at least one compound or group of compounds according to b] comprises a europium salt-containing compound and that the at least one compound or group of compounds according to c] (acceptor group) may comprise allophycocyanine (Grepin et al. (2000), Drug Discovery Today 5, 212).
  • the donor and acceptor groups may be dyes suitable for emissionless energy transfer. It is furthermore possible to use all compounds known to the skilled worker which are suitable for emissionless energy transfer, in particular for fluorescence resonance energy transfer (see, for example, Pope et al. (1999), Drug Discovery Today 4, 350).
  • the signal from the donor component to the acceptor component can be transferred via singlet oxygen.
  • the donor component according to c] can comprise a compound which is able to convert triplet oxygen to singlet oxygen after excitation by a laser wherein the acceptor component according to d] comprises at least one first compound excitable by singlet oxygen and at least one second compound capable of absorbing in an emissionless manner and emitting in the form of fluorescence radiation the energy absorbed by the first compound.
  • the singlet oxygen formed may diffuse from the donor group to the acceptor group and react with the chemiluminescent substances present there.
  • the energy released in the process is transferred to the fluorophores which finally emit the energy in the form of fluorescence radiation which can be detected using a photomultiplier.
  • a precondition for a detectable signal is the spatial proximity of donor and acceptor beads, since singlet oxygen is unstable and decays in aqueous solution. Therefore, the binding components to be used in this method are chosen in such a way that, when the binding event occurs, the distance between the donor and acceptor groups is preferably less than 200 nm.
  • donor group and acceptor group localized on particles the particles preferably having an average diameter of between 150 and 250 nm, particularly preferably of approximately 200 nm.
  • the preferred use of the particles here is such that the final concentration of acceptor group-carrying particles is 1-40 ⁇ g/ml and the final concentration of acceptor group-carrying particles is 1-80 /vg/ml.
  • the binding capacity of the particles may be, for example, from approximately 0.1 nM to 1 nM per ⁇ m/ml particles.
  • the donor group is excited by irradiating at a wavelength of 680 nm, using a laser, and the radiation emitted by the acceptor group can be detected at a wavelength between 520 and 600 nm. According, it is possible, in this case, for the method to be carried out in such a way that donor and acceptor beads carrying donor and acceptor groups are introduced initially.
  • the donor and/or acceptor compound can consist of particles on which donor groups or acceptor groups are localized on. Such particles have preferably an average diameter of approximately 200 nm.
  • the method of the invention for detecting the phosphorylation of a substrate molecule may further comprise in another embodiment the steps of adding at least one test substance and observing how the test substance influences the activity of the protein kinase.
  • the method of the invention is in either case an HTS (high throughput screening)- capable method which may be carried out hom ⁇ genously in the form of a one-step method.
  • the method of the invertion is carried out according to the "mix and measure" principle.
  • the HTS-capable assay systems of the invention are used here preferably for carrying out the above-described methods of the invention.
  • a particular characteristic feature of the methods of the invention is the possibility of monitoring the kinasing of the substrate in solution, since none of the reactants needs to be immobilized on a solid phase. Moreover, it is possible to measure the protein kinases from biological sources directly.
  • Fluorescence is, in theory, the most sensitive analytical technique. As many intra- and intermolecular processes are able to modulate the label emission, it was therefore under consideration very early on as a technique of choice for designing homogeneous assays. Polarization, quenching, time correlation, lifetime variation as well as fluorescence resonance energy transfer (FRET) have been applied to probing molecular interactions. Among these techniques, FRET is of particular interest.
  • the distance Ro (for which transfer is 50% efficient) lies within the 1-7 nm range. Taking advantage of these properties, FRET was used as a spectroscopic ruler to reveal proximity relationships in biological macromolecules.
  • the invention comprises an assay for determining the activity of a G-Protein-Coupled-Receptor specific protein kinase wherein a] a protein kinase is provided, b] a donor bead, which streptavidin is linked to, of an amplified luminescent proximity homogenous assay is provided, c] a substrate molecule, which a biotin molecule is covalently linked to, of the protein kinase from a] is provided, which substrate molecule is phosphorylatable by the protein kinase from a], d] an antibody is provided that specifically recognises the phosphorylated substrate molecule from c] e] an acceptor bead, which is or can be linked to the antibody from d] by means of, e.g., protein A or a suitable secondary antibody, of an amplified luminescent proximity homogenous assay is provided, f] the protein kinase from a], the donor bead from b],
  • the assay for determining the activity of a G-Protein- Coupled-Receptor specific kinase as mentioned before the protein kinase according to a] is selected from the group of ⁇ -Adrenergic-Receptor-Kinase 1 ( ⁇ ARK1] or G- Protein-Coupled-Receptor 2 (GRK2].
  • the assay for determining the activity of a G-Protein- Coupled-Receptor specific kinase as mentioned before the substrate according to c] consists preferably of a G-Protein-Coupled-Receptor which is selected from the group of a muscarinic Acetylcholine-Receptor or an adrenergic alpha 2 Receptor.
  • the assay for determining the activity of a G-Protein- Coupled-Receptor specific kinase as mentioned before the substrate according to c] consists preferably of a peptide being composed of between 10 and 50 amino acids or consists more preferably of a peptide being composed of an amino acid sequence of Seq. ID No. 1.
  • the invention refers to a kit of parts, which kit of parts comprises a] at least one protein kinase, b] at least one donor bead, which streptavidin is linked to, of an amplified luminescent proximity homogenous assay, c] at least one substrate molecule, which a biotin molecule is covalently linked to, of a protein kinase from a] which substrate molecule is phosphorylatable by a protein kinase from a], d] an antibody is provided that specifically recognises the phosphorylated substrate molecule from c] e] at least one acceptor bead, which is or can be linked to the antibody from d] by means of, e.g., protein A or a suitable secondary antibody, of an amplified luminescent proximity homogenous assay is provided.
  • kits of parts may be used simultaneously or one after another in one combined or several distinguished use forms.
  • a use form consists for example of an incubation approach including a vessel for incubation, a liquid for incubation and incubation conditions as temperature or time.
  • the protein kinase according to a] is selected from the group of ⁇ -Adrenergic-Receptor-Kinase1 ( ⁇ ARKI] or G-Protein-Coupled-Receptor-Kinase2 (GRK2].
  • the substrate according to c] consists preferably of a G-Protein-Cou pas-Receptor or more preferably of a G- Protein-Coupled-Receptor which is selected from the group of a muscarinic Acetylcholine-Receptor or an adrenergic alpha2 Receptor.
  • the substrate according to c] consists preferably of a peptide being composed of between 10 and 50 amino acids or consists more preferably of a peptide being composed of an amino acid sequence of Seq. ID No. 1.
  • Such antibody can be linked to the acceptor bead via protein A or a suitable secondary antibody.
  • the invention comprises the manufacturing of a kit of parts which kit of parts has been described as mentioned before wherein a] at least one protein kinase is provided, b] at least one donor bead, which streptavidin is linked to, of an amplified luminescent proximity homogenous assay is provided, c] at least one substrate molecule, which a biotin molecule is covalently linked to, of a protein kinase from a] which substrate molecule is phosphorylatable by a protein kinase from a], is provided, d] an antibody is provided that specifically recognises the phosphorylated substrate molecule from c] e] at least one acceptor bead, which is or can be linked to the antibody from d] by means of, e.g.,
  • the kit of parts as described before can be used for determining the activity of a protein kinase and/or can be used in a screening process which may be organized as High-Throughput-Screening (HTS) for identifying a compound which increases the activity of a protein kinase and/or can be used for identifying a compound which decreases the activity of a protein kinase and/or can be used in a screening process which may be organized as High-Throughput-Screening (HTS] for identifying a compound which decreases the activity of a protein kinase.
  • HTS High-Throughput-Screening
  • the invention comprises a method for identifying a compound, which decreases the activity of a G-Protein-Coupled-Receptor specific protein kinase, wherein a] a protein kinase is provided, b] a donor bead, which streptavidin is linked to, of an amplified luminescent proximity homogenous assay is provided, c] a substrate molecule, which a biotin molecule is covalently linked to, of the protein kinase from a] is provided, which substrate molecule is phosphorylatable by the protein kinase from a] d] an antibody is provided that specifically recognises the phosphorylated substrate molecule from c] e] an acceptor bead, which is or can be linked to the antibody from d] by means of, e.g., protein A or a suitable secondary antibody, of an amplified luminescent proximity homogenous assay is provided, f] a chemical compound is provided, g] the protein is provided
  • a method for identifying a compound which decreases the activity of a G-Protein-Coupled-Receptor specific protein kinase is selected from the group of ⁇ -Adrenergic-Receptor- Kinasel ( ⁇ ARKI] or G-Protein-Coupled-Receptor-Kinase2 (GRK2].
  • a method for identifying a compound which decreases the activity of a G-Protein-Coupled-Receptor specific protein kinase as mentioned before the substrate according to c] consists preferably of a G-Protein-Coupled-Receptor or consists more preferably of a G-Protein-Coupled-Receptor which is selected from the group of a muscarinic Acetylcholine-Receptor or an adrenergic alpha 2-Receptor.
  • a G-Protein-Coupled-Receptor specific protein kinase as mentioned before the substrate according to c] consists preferably of a peptide being composed of between 10 and 50 amino acids or consists more preferably of a peptide being composed of an amino acid sequence of Seq. ID No. 1.
  • the monoclonal antibody E11/19 can be used.
  • Such antibody can be linked to the acceptor bead via protein A or a suitable secondary antibody.
  • the invention comprises a chemical compound having the ability to diminish the activity of a G-Protein-Coupled-Receptor specific kinase which is identified by a method for identifying a compound, which decreases the activity of a G- Protein-Coupled-Receptor as has been described in a former part of this invention.
  • Such compound has preferably a mass of between 0,1 to 50 kDa.
  • Such compound is preferably selected from one of the following groups of a protein, an amino acid, a polysaccharide, a sugar, a polynucleotide, a nucleotide, a fatty acid containing compound, a fat, a fatty acid, a derivative of a fatty acid and or an aromatic carbon compound.
  • Such a compound could be the component of a medicament besides pharmaceutically acceptable expedients for the treatment of a disease which is linked to a disabled phosphorylation of a G-Protein-Coupled-Receptor.
  • the invention comprises in one embodiment a peptide consisting of an amino acid sequence of Seq. ID No. 1.
  • a peptide could be biotinylated.
  • Synthesis of the peptide according to Seq. ID No. 1 carrying or lacking a Biotin-residue can be carried out by a peptide synthesis machine.
  • Such synthesis machines are commercially available e.g. by Perkin Elmer.
  • the peptide according to Seq. ID No. 1 carrying or lacking a Biotin residue can be used as substrate molecule in a protein kinase test.
  • the invention concerns further the use of antibody E11/19 for detecting of a protein or peptide which is phosphorylated.
  • This protein or peptide may encompass or consist of an amino acid according to Seq ID No. 1.
  • Providing of a protein can be performed by a several step approach possibly including cloning of the corresponding gene of the protein in an appropriate expression vector, the transformation of e.g. bacterial cells, cultivation of such cells and expression of the said protein, harvesting of the cells from cultivation medium and the breakdown of the cells, and further isolation of the protein by e.g. ion exchange chromatography, size exclusion chromatography, high pressure liquid chromatography, affinity chromatography, polyacrylamide chromatography or other methods.
  • the person skilled in the art will find appropriate methods for purification and handling of proteins in standard textbooks as in e.g. "Current Protocols in Protein Science - ISBN:0-471- 11184-8 (eds: John E. Coligan, Ben M. Duan, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley Interscience 1995-2003)"
  • Providing a cell includes the preparation, cultivation and further processing of the cell.
  • Cells are provided, for example, by preparing suitable cell material from organs or tissues or by propagating suitable cell lines or microorganisms. Various suitable culture media can be used for cultivation. The cells are maintained at the optimum temperature for the organism. Where appropriate, preservatives, antibiotics, pH indicators, blood serum components, blood serum, auxiliaries or the other substances are added to the growth medium used in each case. Processes for preparation, cultivation and further processing are described in standard textbooks (Example: Basic Cell Culture; Ed. J.M. Davis; IRL Press; 1994).
  • a construct to be expressed in a cell Such a construct consisting of a polynucleotide sequence can be prepared by a skilled worker in a routine manner with the aid of his specialist knowledge. The worker skilled in molecular biology / biochemistry can find the specialist knowledge for this, for example, in "F.M. Ausubel et al.; Current Protocols in Molecular Biology; John Willey & Sons; New York".
  • a vector construct for example is prepared by incorporating a polynucleotide coding for the amino acid sequence of, for example, a GPCR into an expression vector.
  • An expression vector is a vector in which a polynucleotide sequence can be translated in a host cell into a protein.
  • Vectors may be derived from plasmids, viruses or cosmids and must be capable of autonomous replication. They generally contain an origin of replication, cleavage sites for restriction enzymes and marker genes such as, for example, antibiotic resistance genes.
  • the polynucleotide sequence which is to be propagated or which has been introduced from the outside is under the functional control of a promoter.
  • a promoter is a functional polynucleotide sequence of variable length, which is used to control transcription, i.e. synthesis of mRNA of a polynucleotide sequence immediately 3'- of said promoter.
  • the recombinant vector construct comprises an expression vector usable in eucaryotes and/or procaryotes.
  • An expression vector contains a promoter which can be linked functionally to a polynucleotide sequence so that a protein encoded by said polynucleotide sequence is synthesized in an organism, for example a bacterium, fungus or the cell of a eucaryotic cell line.
  • the promoter may be inducible, e.g.
  • Transfection is the introduction of foreign polynucleotide sequences into a host cell and the subsequent propagation of said polynucleotide sequence to any number of identical copies.
  • a cell line is transiently transfected with a recombinant construct by means of routine methods which can be found by the skilled worker in the above mentioned textbook “Current Protocols in Molecular Biology, published by John Wiley & Sons, New York", or in “Sambrook et al.; A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN 0- 87969-309-6". Examples of such routine methods are electroporation, Ca 2+ -phosphate coprecipitation and transfection by means of liposomes.
  • the transfected genes may be expressed in the host cell by Western blotting of cell lysates of transfected cells in combination with an immunological detection method. For this too, the required laboratory protocols can be found by the skilled worker in the manuals mentioned above. Specific antibodies for immunodetection of different GPCR receptors are commercially available.
  • a chemical compound is provided in particular by chemical synthesis or isolation of chemical substances from biological material.
  • Suitable solvents may contain 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. HCI, H 2 SO 4 , formic acid, acetic acid, etc.], bases (e.g. NaOH, etc.], alcohol (e.g. methanol, ethanol, glycerol], detergents (e.g. Na dodecyl sulfate, etc.], organic solvents (e.g. formamide, acetone, dimethyl sulfoxide, etc.] and other components, in particular solubilizers and stabilizers.
  • 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. HCI, H 2 SO
  • the skilled worker is able to bring in contact the components of the assay of the invention by using laboratory routine methods.
  • Contacting may take place, for example, in Erlenmeyer vessels, tubes, Eppendorf vessels or in microtiter plates. Temperature-controlled incubators for which a constant temperature of, for example, 30°C or 37°C and fixed CO 2 or humidity conditions can be set may be used for said contacting. Contacting may in particular also be carried out in laboratory robot devices. Contacting is possible for different periods of time, from a few seconds to minutes and up to several hours. The conditions to be chosen in each case depend on the receptor, the cell line and the chemical compound.
  • the final form of pharmaceutical relates to the final formulation, for example, as tablet, granules, spray, solution, ointment, tincture or other formulation forms.
  • Processing to the final form refers to the preparation of the particular formulation in generally, the daily dose is in the range from 0.3 mg to 100 mg (typically from 3 mg to 50 mg) per day and per Kilogram of body weight, for example, 3-10 mg/kg/day.
  • An intravenous dose may be, for example, in the range from 0.3 mg to 1.0 mg/kg and can most suitably be administered as infusion of from 10 ng to 100 ng per kilogram and per minute.
  • Suitable infusion solutions for these purposes may contain, for example, from 0.1 ng to 10 mg, typically from 1 ng to 10 mg, per milliliter.
  • Single doses may contain, for example, from 1 mg to 10 g of the active substance.
  • ampoules for injections to contain, for example, from 1 mg to 100 mg
  • single-dose formulations which can be administered orally such as, for example, tablets or capsules
  • Detection solution anti-streptavidin antibody (1 :833], streptavidin donor beads (20 ⁇ g/ml], protein-A acceptor beads (40 ⁇ g/ml] in a buffer mixed from 1 part test buffer A containing the kinase, 2 test buffer A containing the kinase substrates and 2 parts test buffer B containing the phosphospecific antibody - 1 ⁇ l compound - + 5 ⁇ l detection solution - seal the plate, incubate overnight at room temperature - read the plate in the Alpha Quest
  • Assay quality parameters were determined on the basis of high and low controls, which were run as internal standards on each assay plate. The following quality parameters are used as a measure to judge the suitability of an assay for high throughput screening.
  • meanhigh control S/B mean ⁇ o control
  • Table 1 Stock solutions
  • Table 2 Buffers and reagents
  • Fig. 1 Assay validation; ATP dependence; the apparent Km value was determined at 4 ⁇ M
  • Fig. 2 Stability of the reaction mix; reagents are stable for more than 3 hours.
  • Fig. 3 DMSO dependence of the assay; the assay tolerates up to 5% DMSO
  • Fig. 4 Dose-response curve for heparin; the IC 50 was determined as 50 nM
  • Fig. 5 Amino acid sequence according to Seq. Id No.1
  • Fig. 6 Amino acid sequence including the ⁇ ARK-1 Sequence according to Seq. ID No. 2

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Abstract

La présente invention se rapporte à un dosage qui permet de déterminer l'activité d'une protéine kinase spécifique du récepteur couplé à la protéine G, et ce à l'aide d'un dosage homogène de proximité par luminescence amplifié.
EP04764711A 2003-09-11 2004-09-02 Criblage a haut rendement et sensibilite elevee pour mesurer l'activite kinase du recepteur beta-adrenergique et pour determiner l'impact sur cette activite de substances a tester Withdrawn EP1664333A2 (fr)

Priority Applications (1)

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EP04764711A EP1664333A2 (fr) 2003-09-11 2004-09-02 Criblage a haut rendement et sensibilite elevee pour mesurer l'activite kinase du recepteur beta-adrenergique et pour determiner l'impact sur cette activite de substances a tester

Applications Claiming Priority (3)

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EP03020429A EP1514944A1 (fr) 2003-09-11 2003-09-11 Criblage à haut rendement et sensibilité élevée pour mesurer l'activité kinase du récepteur béta-adrénergique et pour déterminer l'impact sur cette activité de substances à tester
EP04764711A EP1664333A2 (fr) 2003-09-11 2004-09-02 Criblage a haut rendement et sensibilite elevee pour mesurer l'activite kinase du recepteur beta-adrenergique et pour determiner l'impact sur cette activite de substances a tester
PCT/EP2004/009751 WO2005024426A2 (fr) 2003-09-11 2004-09-02 Dosage haut debit a grande sensibilite permettant de mesurer l'activite d'une kinase specifique du recepteur $g(b)-adrenergique et de determiner l'influence de substances d'essai sur une telle activite

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EP1664333A2 true EP1664333A2 (fr) 2006-06-07

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EP04764711A Withdrawn EP1664333A2 (fr) 2003-09-11 2004-09-02 Criblage a haut rendement et sensibilite elevee pour mesurer l'activite kinase du recepteur beta-adrenergique et pour determiner l'impact sur cette activite de substances a tester

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