EP1627073A2 - Utilisation d'un polypeptide biotinyle pour la determination de l'activite des enzymes phosphorylantes de proteines - Google Patents

Utilisation d'un polypeptide biotinyle pour la determination de l'activite des enzymes phosphorylantes de proteines

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Publication number
EP1627073A2
EP1627073A2 EP04729646A EP04729646A EP1627073A2 EP 1627073 A2 EP1627073 A2 EP 1627073A2 EP 04729646 A EP04729646 A EP 04729646A EP 04729646 A EP04729646 A EP 04729646A EP 1627073 A2 EP1627073 A2 EP 1627073A2
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EP
European Patent Office
Prior art keywords
polypeptide
enzyme
ability
irs
derivative
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.)
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EP04729646A
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German (de)
English (en)
Inventor
Norbert Tennagels
Aimo Kannt
Harald Thuering
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Sanofi Aventis Deutschland GmbH
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Sanofi Aventis Deutschland GmbH
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Publication of EP1627073A2 publication Critical patent/EP1627073A2/fr
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates to the use of a polypeptide to determine the ability of an enzyme to modulate the phosphorylation state of the polypeptide. Further aspects of the invention relate to a method for determining such an activity and to a method for identifying substances which modify this ability of the enzyme.
  • Insulin is a peptide hormone that affects a large number of growth and metabolic pathways by binding to the insulin receptor, thereby activating its intrinsic tyrosine kinase. This event leads to the phosphorylation of a variety of proteins that can bind to the insulin receptor (IR) on specific tyrosine residues.
  • IR insulin receptor
  • the family of insulin receptor substrate (IRS) proteins also belongs to the proteins phosphorylated in this way.
  • Insulin receptor substrate 1 is a cellular protein which can be phosphorylated by a large number of protein kinases (tyrosine-specific or serine / threonine-specific protein kinases) on tyrosine and / or serine residues and or threonine residues. Depending on the enzyme, different tyrosine or serine / threonine residues will presumably be phosphorylated specifically.
  • protein kinases tyrosine-specific or serine / threonine-specific protein kinases
  • IGF-1 receptor White 2002
  • JAK 1/2 Thife et al.
  • IRS-1 is also known to be produced by serine / threonine kinases such as kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c-Jun NH (2) - terminal kinase (JNK, Aguirre et al. 2000) Protein kinase A (Sun et al. 1991) , Mitogen activated protein kinase (Mothe et al. 1996), protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al.
  • serine / threonine kinases such as kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c-Jun NH (2) - terminal kinase (
  • IRS molecules are key molecules in the insulin signal transduction pathway and play a central role in the maintenance of cellular functions such as growth, survival and metabolism.
  • Phosphorylated IRS proteins serve as "docking" proteins with a variety of docking sites for the insulin receptor and a complex network of intracellular signaling molecules with so-called Signal Recognition Complex (SRC) homology 2 domains (SH2 Domains).
  • SRC Signal Recognition Complex
  • IRS belongs to a group of phosphoproteins from 160 to 185 kDA in size that serve as a substrate for the insulin receptor.
  • IRS-1, IRS-2, IRS-3 and IRS-4 are known. They differ in tissue distribution, subcellular localization, development-specific expression, type of binding to the insulin receptor and the type of SH2 proteins with which they interact.
  • the four members of the IRS family are very similar in their basic protein structure: All have an amino (N) -terminal Plextrin homology domain (PH domain) that binds to membrane phospholipids, a phosphotyrosine binding domain (PTB domain) that is directly connects carboxy (C) terminal to the PH domain and is involved in the detection of the Asp-Pro-Glu phosphotyrosine (NPEpY) sequence which beta-insulin receptor in the juxta-membrane region. Subunit is localized. Furthermore, they have a somewhat less conserved C-terminal part, which has various potential tyrosine phosphorylation motifs to which special SH2 domain-containing proteins can bind.
  • NPEpY Asp-Pro-Glu phosphotyrosine
  • IRS-1 contains 21 possible, tyrosine phosphorylation sites, some of which are located in Aminpklaresequenzmotiven that can bind to the SH2 Domjänen proteins. IRS-1 also contains 30 potential serine / threonine phosphorylation sites in motifs that can be recognized by various kinases such as kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c- Jun NH (2) terminal kinase (JNK, Aguirre et al. 2000) Protein kinase A (Sun et al. 1991), Mitogen activated protein kinase (Mothe et al.
  • Protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al. 1993), glycogen synthase kinase beta (Eldar-Finkelmann et al. 1997), AMP activated kinase (Jakobsen et al. 2001) or phosphoinositol 3 kinase (PI3 Kinase, Freund et al. 1995).
  • Inhibitory effects on the insulin receptor signaling pathway can be at least partly due to the recently discovered role of serine / threonine phosphorylation of IRS-1 explained, which is associated with a deterioration in the interaction with the insulin receptor and / or a reduction in the tyrosine phosphorylation of IRS-1 and / or a deterioration in the interaction with subsequent signal proteins which can bind to tyrosine phosphorylated IRS-1 5 (for For an overview, see White 2002).
  • kinases for example kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al.
  • JNK c-Jun NH (2) terminal kinase
  • JNK c-Jun NH (2) terminal kinase
  • protein kinase A (Sun et al. 1991), mitogen-activated protein kinase (Mothe et al. 1996), protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al. 1993)
  • IRS-1 15 residues in some studies are directly related to the reduced tyrosine phosphorylation by the insulin receptor (Le Marchand-Brustel 1999)).
  • the sequences of IRS-1, 2, 3 and 4 are publicly available.
  • the coding polynucleotide sequences and the associated protein sequences of these genes are under the numbers NM_005544 (IRS-1 hs), XM: 007095 (IRS-2 hs), NM: 032074
  • IRS-1 20 (IRS-3 rat), NM: 003604 (IRS-4 hs) available from the NCBI Nucleotide Database.
  • NCBI is the National Center for Biotechnology Information (postal address: National Center for Biotechnology Information, National Library of Medicine, Building 38A, Bethesda, MD 20894, USA; web address: www.ncbi.nhm.nih.gov).
  • the cloning of the IRS-1 gene was described, inter alia, in Araki et al. 1993 and Sieffle et. al,
  • IRS-2 to 4 was carried out by Araki et al 1994, Lavan et al. 1997a and Lavan et al. 1997bbeprim.
  • IRS-1 fragments for example a fragment of IRS-1 (amino acid 516-777) and insulin receptor, IGF-1 receptor or recombinant insulin receptor kinase
  • a fragment of IRS-1 amino acid 516-777
  • insulin receptor IGF-1 receptor
  • recombinant insulin receptor kinase Incubation with radioactively labeled ATP, dropping of the substrate onto a positively charged membrane (nitrocellulose or similar material), Washing and detection of the bound radioactively labeled substrate by means of autoradiography or measurement of the radioactive radiation.
  • the disadvantage of the radioactive test methods described above is obvious, since the use of radioactivity entails considerable dangers, is very cost-intensive and is therefore not particularly suitable for high-throughput methods (HTS methods).
  • the disadvantage of the methods described above, which are based on the use of short peptides, is that these peptides have unfavorable kinetic constants (Vmax, Km) and, moreover, the spatial structure in the case of peptides is very different from that of the physiological enzyme substrates.
  • the object of the invention is therefore to provide a possibility for determining the activity of protein phosphorylating and / or dephosphorylating enzymes which does not have the disadvantages mentioned above.
  • This object is achieved by using a polypeptide (Def GGs to the peptide) to determine the ability of an enzyme, a functional fragment or derivative thereof to modulate the phosphorylation status of a polypeptide, characterized in that the polypeptide is biotinylated.
  • the invention is based on the results of the inventors, who surprisingly showed that even with polypeptides or full-length proteins there was no steric hindrance to the binding of biotin by streptavidin and that the biotinylation did not interfere with the phosphorylation of the substrate under investigation by kinases.
  • polypeptide means a molecule containing amino acids linked by peptide bonds, which has at least 50 amino acids linked linearly in this way. Shorter molecules of this type are called peptides.
  • protein refers to molecules that comprise at least one polypeptide chain, but can also consist of several polypeptide chains that are associated or linked with one another. The term protein thus includes the term polypeptide. According to a preferred embodiment of the various aspects of the present invention, the polypeptide has a length of 50 amino acids and more, preferably 50-300.
  • the polypeptide has a size of 1 kda and more, preferably 1 to 100 kDa and particularly preferably 10 to 50 kDa.
  • substrate of an enzyme is understood to mean any molecule that is suitable for being modified by the enzyme.
  • natural substrates are molecules which are designed in such a way as they occur in nature in a physiological or pathological context and are capable of being modified by the enzyme in question.
  • the modulation of the state of phosphorylation by the enzyme denotes the type of modification of a substrate by an enzyme in which at least one phosphate group is transferred to or removed from the substrate.
  • the enzymes relevant to the present invention therefore have the ability to catalyze one and / or the other reaction. They therefore have at least this ability of the kinases and / or the phosphatases, but can also have other enzymatic properties (e.g. protease properties, etc.).
  • the various enzyme categories and their properties are well known to the person skilled in the art.
  • a functional fragment of an enzyme is any fragment of the enzyme (that is, a molecule that is reduced or shortened compared to the naturally occurring form) that still has the ability to modulate the phosphorylation state of at least one polypeptide.
  • the term "functional derivative" of an enzyme encompasses any type of modification of the enzyme compared to the naturally occurring form, which does not constitute a shortening, the derivative of the enzyme still having the ability to modulate the phosphorylation state of at least one polypeptide.
  • the present invention also refers to functional derivatives of fragments of enzymes that can modulate the phosphorylation state of at least one polypeptide. The ability of the enzyme to modulate the phosphorylation state of the polypeptide can be determined both qualitatively and quantitatively (ie as a quantifiable measurement).
  • the use according to the invention has the advantage that the results obtained in this way are more meaningful due to the length of the substrates used, since they assume a tertiary structure that more closely corresponds to the physiological conditions.
  • the polypeptides used have good kinetic constants (for example with IRS-1: Km 19 ⁇ M: compared to peptides:> 200 ⁇ M see Sieffle et al. 1995) and it is in the analysis of substrates with several phosphorylation sites only one substrate is necessary with which, for example the ability of various enzymes can also be determined.
  • a preferred embodiment relates to a use in which the ability of an enzyme to phosphorylate the polypeptide is determined.
  • Particularly useful types of enzymes with kinase activity for the various aspects of the present invention relate to serine / threonine or tyrosine kinases.
  • Particularly suitable examples of kinases include u. a.
  • the use according to the invention is also suitable for determining the ability of an enzyme to dephosphorylate the polypeptide.
  • Another aspect of the invention relates to a method for determining the ability of an enzyme, a functional fragment or derivative thereof to modulate the phosphorylation status of a biotinylated polypeptide.
  • Suitable methods for determining the degree of phosphorylation of biotinylated polypeptides relate, for example, to methods which are known to be suitable for determining the degree of phosphorylation of short peptides. These are familiar to the person skilled in the art.
  • the method according to the invention relates to a method in which the ability of an enzyme, a functional fragment or derivative thereof to phosphorylate the polypeptide is determined by the following steps: a) contacting the enzyme or functional fragment or derivative with the biotinylated polypeptide and starting the phosphorylation reaction in a suitable reaction mixture, b) contacting the reaction mixture with an a carrier-coupled agent capable of binding to the biotinylated polypeptide, c) determining the phosphorylation state of the polypeptide bound to the agent.
  • a further preferred embodiment of the invention relates to a method for determining the ability of an enzyme, a functional fragment or derivative thereof to dephosphorylate the polypeptide with the steps a) bringing the enzyme or functional fragment or derivative into contact with the biotinylated polypeptide, which has at least one Has phosphate residue and start the phosphoryiation reaction in a suitable
  • Reaction batch b) contacting the reaction batch with an agent coupled to a carrier capable of binding the biotinylated polypeptide, c) determining the phosphorylation state of the polypeptide bound to the agent.
  • the agent can be any type of molecule or supramolecular assembly (e.g. body or device) that is suitable for binding the biotinylated polypeptide.
  • the binding can take place to the biotin portion or to the polypeptide itself, with one binding to the polypeptide itself
  • binding dependent on the phosphorylation state is preferred (eg binding only in the phosphorylated or unphosphorylated state with reference to individual or more phosphorylation sites).
  • Preferred embodiments of the agent therefore relate to streptavidin or phosphospecific antibodies (ie antibodies which recognize the phosphorylation of certain residues on the polypeptide and can bind specifically to the polypeptide phosphorylated there).
  • the reaction mixture used in the various aspects of the invention can be biochemical (ie in vitro) or cellular.
  • the composition of biochemical approaches depends on the requirements of the enzyme to be investigated, but suitable constituents and compositions, for example ATP, a buffer for setting a desired pH environment and a desired salt concentration for ensuring the enzyme activity are known to the person skilled in the art.
  • enzyme and or polypeptide can be present recombinantly and / or as a molecule partially or completely purified from natural sources and / or in the form of extracts from biological material, in particular cell or tissue extracts.
  • Biological material can include, among others: the cells of a tissue or organ (e.g. brain, blood, liver, spleen, kidney, heart, blood vessels), preferably those of a vertebrate including humans, or cells from a cell culture.
  • Cells used in the context of the invention include all types of cells, e.g. eukaryotic or prokaryotic unicellular organisms (such as bacteria, e.g. E. coli or yeasts, e.g. S. pombe or s. cerevisiae) or cell lines derived from multicellular organisms (such as HeLA, COS, NIH-3T3, CHO, etc.) , Mammalian cell lines are preferred.
  • Cells from a tissue association or organ of a vertebrate, including humans, can be obtained using common techniques such as “blood collection, tissue puncture or surgical techniques.
  • blood collection, tissue puncture or surgical techniques The production of such recombinant molecules, the purification of naturally occurring molecules from cells or tissues and the production of cell or tissue extracts is well known to the person skilled in the art (see also examples of the standard literature listed below).
  • Cellular systems suitable for use in the various aspects are also known to the person skilled in the art and preferably comprise isolated cells which originally come from tissue associations (preferably from vertebrates, particularly preferably mammals and in particular humans), particularly preferably in the form of cultivated cell lines; they also include unicellular organisms (eukaryotes or prokaryotes), such as yeast or bacterial cells, especially in the form of cultivated strains.
  • Carriers can be all types of molecules or supramolecular assemblies (eg bodies or devices) which are suitable for removing or labeling the peptide coupled to them via the biotin-streptavidin interaction from the reaction mixture.
  • Suitable devices are, for example, membranes, plates or bodies of various shapes (generally referred to as beads in the present case), made of various materials which are well known in the prior art.
  • the type of carrier depends on the process objective (e.g. diagnostic, drug discovery or discovery of new interaction partners) and the type of detection, the selection of suitable carriers is within the range of expert knowledge.
  • radioactively labeled ⁇ 32P-ATP is added to the reaction mixture, and the phosphorylation state is determined by measuring the radioactivity remaining on the support, preferably the membrane or plate, after carrying out at least one washing step.
  • the components of the reaction mixture, including free radioactivity, which are not bound to the streptavidin can simply be removed, so that the phosphorylation state of the polypeptide can easily be determined on the basis of the radioactivity immobilized on the support.
  • Streptavidin is particularly suitable in this case as a means of binding the biotinylated polypeptide.
  • an antibody is added to the reaction mixture, which is able to bind specifically to the phosphorylated polypeptide.
  • the antibody can both be the agent itself and can be added in addition to the agent (in which case it is then preferably not a phosphospecific antibody and particularly preferably streptavidin).
  • the phosphorylation state is determined here by determining the amount of the antibody bound to the polypeptide. Suitable measures for labeling and detection of the antibody are known to the person skilled in the art.
  • appropriately labeled first antibodies can be used that are directly detectable, or appropriately labeled second antibodies directed against the FC (crystalizing fragment) portion of the first antibody can be used, which increases the specificity of the detection.
  • Suitable labels for such antibodies are also known in the prior art and include, for example, enzymatic labels such as CIP (Calf intestinal phosphatase) or HRP (Horseraddish Peroxidase), fluorescent molecules which, when excited by irradiation with light of a certain wavelength, generate a detectable signal such as Texas Red, Cy3, FITC (fluorescein isothiocyanate), or known fluorescent proteins.
  • CIP Calf intestinal phosphatase
  • HRP Haseraddish Peroxidase
  • fluorescent molecules which, when excited by irradiation with light of a certain wavelength, generate a detectable signal such as Texas Red, Cy3, FITC (fluorescein isothiocyanate), or known fluorescent proteins.
  • the selection of suitable markings also corresponds to the professional ability.
  • first and second antibodies Suitable labeled or unlabeled first and second antibodies and their production are known in the art, moreover such antibodies are commercially available from various suppliers.
  • First and second antibodies are available, for example, from Becton Dickinson, Pharmacia or Santa Cruz Biotech.
  • the amount of the antibody bound to the polypeptide is determined by determining the amount of the antibody remaining on the support, preferably the membrane or plate, after carrying out at least one washing step.
  • the carrier coupled to the agent is a first carrier that includes a first signal generator and the polypeptide is coupled to a second carrier that includes a second signal generator, the two signal generators being able to generate a detectable signal when they are are in close proximity to each other and the determination of the phosphorylation state using the
  • the carriers are preferably beads.
  • the agent here is preferably a phosphospecific antibody.
  • the carrier can be directly or indirectly connected to the antibody, preferably indirectly through ProteinA, which is coupled to the carrier.
  • the second carrier can be bound directly or indirectly to the polypeptide, preferably indirectly through the biotin portion of the biotinylated polypeptide; this is preferably done via streptavidin coupled to the carrier.
  • a signal generator can be any type of agent or molecule that is suitable for generating detectable signals; Examples include fluorophores that emit light upon excitation by energy, which can be detected directly or after signal amplification by suitable means known in the art.
  • the signal generators are selected such that a signal is only generated when the agent (ie preferably the phosphospecific antibody) interacts directly with the polypeptide.
  • Suitable carriers and signal generators for example in the form of the ALPHAScreen TM or LANCE TM, Perkin-Elmer Life Sciences; HTRF TM, CIS Bio International) are known.
  • the immediate proximity of the carriers to each other is crucial for signal generation. It is therefore very surprising that this type of method is suitable for use in conjunction with polypeptides, although these are significantly larger than the peptides used in the prior art.
  • the polypeptide is preferably the natural substrate of the enzyme, preferably in an unabridged length.
  • Particularly suitable polypeptides include all substrates of the insulin receptor kinase.
  • Particularly preferred polypeptides are insulin receptor substrate '(IRS) family, preferably IRS-1, 2, 3 or 4 and particularly preferably IRS-1, or functional fragments or derivatives thereof. That is fragments or derivatives (or derivatives of fragments) which have the ability to be phosphorylated by the insulin receptor. It is further preferred if the IRS is human IRS.
  • IRS-1 in particular human IRS-1 with the sequence according to SEQ ID No.1, is particularly preferred in the context of the various aspects of the present invention, human IRS-1 encoded by the sequence according to SEQ ID No.2.
  • the aforementioned polypeptides are particularly suitable for determining the ability of the insulin receptor to phosphorylate them.
  • a preferred IRS-1 fragment is a polypeptide with the amino acid sequence according to SEQ ID No.3.
  • the various aspects of the invention can be used at different levels. Their use is particularly useful in the identification of Substances that modify the ability of the enzyme or functional fragment or derivative thereof to modulate the phosphorylation state of the polypeptide.
  • Suitable analytical methods or systems which measure the activity or the concentration or amount or specific target molecules of the body (so-called “targets”, in this case the phosphorylation state of the polypeptide), as parameters of the effectiveness of potential active substances, are in the prior art
  • targets in this case the phosphorylation state of the polypeptide
  • these can be, for example, in vitro assays, that is to say biochemical assays with isolated or partially isolated components, which are put together to form a reaction mixture, by means of which the effectiveness of potential active substances can be measured.
  • these can also be cellular test systems (assays) , in which the activity of the target protein (in this case the enzyme) and the effectiveness of potential active substances on the activity of this target molecule in the cellular environment can be determined.
  • An assay is any type of analytical method that can be used to monitor a biological process.
  • Molecular processes and signal cascades which represent parts of physiological metabolic pathways and control mechanisms, but also pathological states, are conventionally simulated in cellular or biochemical systems. The pharmacological activity of an active substance can then be determined on the basis of its ability to intervene in these pathways and mechanisms.
  • the assay For use in the context of drug binding, in particular high-throughput screening for drugs, the assay must be reproducible and is preferably also scalable and robust (i.e. not very sensitive to external influences).
  • the assay should preferably be suitable for high throughput screening of chemical substances for their ability to affect the activity of target molecules.
  • the type of assay depends, among other things, on the type of target molecule used (eg exact type or type of basic biochemical molecule, eg polypeptide or polynucleotide) and the "read out", ie the parameters on the basis of which the activity of the target molecule is determined, from.
  • Various types of assays are known in the prior art and for the most part are also commercially available from commercial suppliers.
  • Assays suitable for measuring the interaction of two binding partners include, for example, radioisotopic or fluorescent assays, for example fluorescence polarization assays, such as those commercially available from Panvera, Perkin-Elmer Life Sciences (NEN, LANCE TM, AlphaScreen TM) or CIS Bio International (HTRF TM).
  • Other examples of assays include cellular assays in which a cell line expresses stably (inducible or constitutive; chromosomal or episomal) or transiently a recombinant protein as desired.
  • These assays include, for example, reporter gene assays in which the regulation of a specific promoter or the regulation of a signal transduction path or a member of a signal transduction cascade is measured on the basis of the activity of a reporter enzyme whose expression is under the control of the promoter in question.
  • reporter gene assays in which the regulation of a specific promoter or the regulation of a signal transduction path or a member of a signal transduction cascade is measured on the basis of the activity of a reporter enzyme whose expression is under the control of the promoter in question.
  • Suitable reporter enzymes are generally known to the person skilled in the art and include fireflies luciferase, Renilla luciferase (both commercially available, for example, from Packard Reagents), ⁇ -galactosidase, etc.
  • the selection of suitable cell lines is known to the person skilled in the art and depends, inter alia, on the aim of the assay or read out ". These are usually cell lines that are easy to cultivate and transfect, such as HeLA, COS, CHO or NIH-3T3 cells.
  • fluorescence polarization are suitable for measuring protein phosphorylation or kinase activity
  • HTRF TM Homogeneous Time Resolved Fluorescence
  • LANCE TM Assays Perkin-Elmer Life Sciences
  • ALPHAScreen TM Amplified Luminescent Proximity Homogeneous Assay
  • present invention particularly expedient measurement of kinase activity using ALPHAScreen TM from Perkin-Elmer Li fe sciences is carried out, for example, in that the kinase to be investigated phosphorylates a biotinylated peptide in a biochemical approach in the presence of ATP.
  • the phosphorylated Peptide is then bound by a specific anti-phospho antibody to which protein A-conjugated acceptor beads or with suitable second antibodies are coupled.
  • protein A-conjugated acceptor beads or with suitable second antibodies are coupled.
  • streptavidin-coupled donor beads that bind the biotin portion of the peptide.
  • acceptor and donor beads come in close proximity, which sets in motion a cascade of chemical reactions that generate a highly amplified, detectable luminescence signal:
  • Laser excitation stimulates a photosensitizer in the donor bead to put ambient oxygen into a singlet status , The singlet oxygen then diffuses to the acceptor bead, where it excites a thioxane derivative, which emits chemiluminescence with a wavelength of 370 nm, which in turn stimulates further fluorophores in the acceptor bead to luminesce light with wavelengths from 520 to 620 nm. Since the singlet oxygen excites the fluorophores only when the donor and acceptor beads are in close proximity, detectable signals are only generated.
  • Other types of assays and other types of "read outs" are also well known to the person skilled in the art.
  • the modification of the modulation can mean an inhibition or activation of the modulation by the enzyme.
  • the type of modification includes all possible influences that ultimately have an effect on the enzyme-catalyzed phosphorylation state of the polypeptide, such as the modification of the enzyme-substrate interaction or the modification of the catalytic activity of the enzyme, but also (preferably in the analysis by means of cellular reaction approaches ) modification of enzyme expression, etc.
  • Another aspect of the invention relates to a method for identifying substances which modify the ability of an enzyme or functional fragment or derivative thereof to modulate the phosphorylation state of a polypeptide with the steps a) determining the ability of the enzyme or functional fragment or derivative from modulating the phosphorylation state of the polypeptide according to one of the above-mentioned methods according to the invention, the substance to be tested not being added to the reaction mixture, b) determining the ability of the enzyme or functional fragment or. Derivative of modulating the phosphorylation state of the polypeptide according to one of the methods according to the invention described above, the substance to be tested being added to the reaction mixture, c) comparing the ability according to a) with that according to b).
  • the methods according to the invention are particularly suitable for the identification of pharmacologically active substances for the treatment of non-insulin-dependent diabetes mellitus (NIDDM), in oncology (IGFRK) or for the treatment of inflammatory processes (IKK kinase).
  • NIDDM non-insulin-dependent diabetes mellitus
  • IGFRK in oncology
  • IKK kinase inflammatory processes
  • the fragment contains five potential tyrosine phosphorylation sites, which are highlighted in FIG. 3 and subsequently together with their
  • Serins 612, 632, 662 and 731 which represent four possible serine kinase phosphorylation sites in YMXMSP motifs, are located near the tyrosine phosphorylation sites of the insulin receptor, which are housed in binding sites for SH2 somenas.
  • the mutation of these serine residues to alanine leads to an increase in the IRS-1 -mediated activity of the phosphatidyl-insositol trisphosphate kinase (PI3K), (Mothe et al. 1996), which indicates that they have an inhibitory function.
  • PI3K phosphatidyl-insositol trisphosphate kinase
  • the 262 amino acid domain D516-P777 (HLRS-1- p30) of human IRS-1 was first expressed in E-coli as described in Siemeister et al., 1995.
  • the expression vectors were thereby inserted by inserting the polynucleotide with the sequence according to SEQ ID No. 10 (cDNA sequence of HLRS-1-p30) into the plasmid pET3d (commercially available under order number 69421 from Novagen), prepared by conventional methods.
  • the empty vector was first digested with the enzymes Ncol (commercially available from Röche Diagnostics GmbH Mannheim under order number 835315) and BamHI (commercially available from Röche Diagnostics GmbH Mannheim under order number 656275) under standard conditions and using spin columns (commercially available from Qiagen, Hilden cleaned under order number 28104).
  • Ncol commercially available from Röche Diagnostics GmbH Mannheim under order number 835315
  • BamHI commercially available from Röche Diagnostics GmbH Mannheim under order number 656275
  • spin columns commercially available from Qiagen, Hilden cleaned under order number 28104.
  • WGA-IR wheat germ lectin affinity chromatographically purified insulin receptor from rat liver
  • WGA-IR wheat germ lectin affinity chromatographically purified insulin receptor from rat liver
  • human insulin for example commercially available from Sigma under order number 14
  • biotinylated IRS fragment for 10 minutes at 4 ° C in 50 mM Tris buffer, pH 7.4, 8 mM MgCl2, 2 mM MnCl2, followed by a 30 minute incubation after addition of ATP (final concentration 50 ⁇ M) at 30 ° C.
  • the reaction was then stopped by adding EDTA to a final concentration of 20 mM and the phosphorylation of IRS-1 was detected by using a p-Tyr-specific antibody directly coupled to the acceptor (commercially available from Perkin-Elmer Life Sciences under order number 6760601 C), which resulted in the readout shown in FIG. With this method, the EC50 for insulin could be determined to be 10 nM.
  • ALPHAScreen TM Phosphorylation of biotinylated IRS-1 fragment by PKC and recombinant insulin receptor kinase.
  • Perkin-Elmer Life Sciences' ALPHAScreen TM enables detection of the interaction between the phosphorylated IRS-1 fragment and antibodies that recognize phosphorylated serine or tyrosine residues (p-Ser / p-Tyr antibody).
  • the biotinylated IRS-1 is bound to the streptavidin donor and the antibody by acceptor-coupled protein A or a suitable second antibody bound to the acceptor.
  • acceptor-coupled protein A or a suitable second antibody bound to the acceptor When an interaction takes place, the acceptor gets and stays in the immediate vicinity of the donor, so that singlet oxygen atoms, which are generated by the donor can reach chemiluninescent groups in the acceptor bead by diffusion, which ultimately results in the emission of detectable light.
  • the light intensities (the so-called “readout”) generated in the aforementioned assay in the form of bar graphs in FIGS. 5 A and B were obtained after 30 minutes incubation of IRS-1 with protein kinase C and ATP and subsequent addition of p-Ser antibodies (commercial available from Biosource, Belgium under order number 44-550) and further incubation for 120 minutes detected and quantified by measurement with a Perkin-Elmer Fusion or AlphaQuest instrument.
  • the comparison of the light intensities generated in the presence and absence of PKC is shown in Figure 11A. In the experiment, the result of which is shown in FIG.
  • recombinant insulin receptor kinase (IRK, amino acid 941- (1343, NCBI accession number NM_000208) was incubated with polyiysin for 10 minutes at 30 ° C. in 50 mM Tris buffer, pH 7 , 4, 8 mM MgCl 2, 50 ⁇ M ATP reaction buffer activated and then the IRK substrate IRS added, followed by a 30 minute incubation ion at 30 ° C. Phosphorylation of IRS-1 was detected using a p-Tyr specific antibody directly coupled to the acceptor (commercially available from Perkin-Elmer Life Sciences under order number 6760601 C), resulting in the readout shown in Figure 11B.
  • IRS insulin receptor kinase
  • biotinylated polypeptides can be phosphorylated by kinases. This was demonstrated using a 28 kDA fragment of hlRS-1, which in the biotinylated state can be phosphorylated by the serine kinase PKC ⁇ and by the tyrosine kinase of the insulin receptor Detection by phosphospecific antibodies was also successful without a steric hindrance due to the size of the polypeptide in connection with the biotin residue, which interfered with the detection reaction, thereby generating a homogeneous assay system based on the principle of the ALPHAScreens TM
  • the phosphorylation state of polypeptides can be determined using the purification and detection techniques possible through biotinylation.This assay principle was applied here for the first time to a protein fragment the size of a polypeptide (more precisely 28kDa) enables the improved search for pharmacologically active substances which interact with the
  • the readout was also non-radioactive but luminescent, which is an advantage for use in high throughput screening (HTS) procedures.
  • the assay shown here can thus be used for the HTS of all enzymes modulating the phosphorylation status of polypeptides and proteins, such as kinases and phosphatases, for identifying novel active substances or for verifying known active substances. It is also suitable for other processes, such as the aforementioned processes for the search for new enzymes which phosphorylate certain polypeptides, for example new IRS-1 phosphorylating kinases in whole cell lysates.
  • IRS 1 - IRS 4 Protein sequence from IRS 1 - IRS 4 (SEQ ID No. 1 to 4).
  • the sequence access numbers (NCBI Protein Database) of the four family members are NM_005544 (IRS-1 hs),: M: 007095 (IRS-2 hs), NM: 032074 (IRS-3 hs), NM_003604 (IRS-4 hs).
  • IRS 1 - IRS 4 Coding DNA sequence from IRS 1 - IRS 4 (SEQ ID No. 5 to 8).
  • sequence access numbers NCBI nucleotide database of the four family members are NM_005544 (IRS-1 hs),: M: 007095 (IRS-2 hs), NM: 032074 (IRS-3 hs), NM_003604 (IRS-4 hs).
  • FIG. 3 The 262 amino acid domain of the IRS-1 protein (hIRS-1-p30), which was used for the present studies. Serins 612, 632, 662 and 731 are underlined. YXXM tyrosine phosphorylation motifs are shown in bold.
  • FIG. 4 results of the ALPHAScreen using insulin receptor purified by wheat germ lectin affinity chromatography
  • Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate.
  • IRS-1 insulin receptor substrates IRS-1
  • IRS-1 insulin receptor substrate-1
  • the PI3-kinase serine kinase phosphorylates its p85 subunit and IRS-1 in PI3-kinase / IRS-1 complexes.
  • Human skeletal muscle insulin receptor substrate-1 Characterization of the cDNA, gene, and chromosomal localization.
  • the 60-kDa phosphotyrosine protein in insulin-treated adipocytes is a new member of the insulin receptor Substrate family. J. Biol. Chem. 272, 11439-11443
  • a novel 160-kDa phosphotyrosine protein in insulin-treated embryonic kidney cells is a new member of the insulin receptor Substrate family. J. Biol. Chem. 272, 21403-21407 De Fea K, Roth RA. (1997)
  • Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612 Biochemistry 36, 12939-12947 Standard literature for laboratory methods

Abstract

L'invention concerne l'utilisation d'un polypeptide visant à déterminer la faculté d'une enzyme, d'un fragment fonctionnel ou d'un dérivé de cette enzyme, à moduler l'état de phosphorylation du polypeptide. L'invention est caractérisée en ce que ledit polypeptide est biotinylé.
EP04729646A 2003-05-22 2004-04-27 Utilisation d'un polypeptide biotinyle pour la determination de l'activite des enzymes phosphorylantes de proteines Withdrawn EP1627073A2 (fr)

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RU2005140091A (ru) 2006-05-10
IL172008A (en) 2011-12-29
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AU2004241327B2 (en) 2010-03-25
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US7732151B2 (en) 2010-06-08
RU2395813C2 (ru) 2010-07-27
US20080020399A1 (en) 2008-01-24
MXPA05012521A (es) 2006-05-25
HK1092840A1 (en) 2007-02-16
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