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EP1466009A2 - Highly sensitive and continuous protein-tyrosine-phosphatase (ptpase) test using 6.8 difluoro-4-methyl-umbelliferylphosphate - Google Patents

Highly sensitive and continuous protein-tyrosine-phosphatase (ptpase) test using 6.8 difluoro-4-methyl-umbelliferylphosphate

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Publication number
EP1466009A2
EP1466009A2 EP02796734A EP02796734A EP1466009A2 EP 1466009 A2 EP1466009 A2 EP 1466009A2 EP 02796734 A EP02796734 A EP 02796734A EP 02796734 A EP02796734 A EP 02796734A EP 1466009 A2 EP1466009 A2 EP 1466009A2
Authority
EP
European Patent Office
Prior art keywords
biological material
compound
difluoro
difmup
protein tyrosine
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Withdrawn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
EP02796734A
Other languages
German (de)
French (fr)
Inventor
Stefan Welte
Norbert Tennagels
Stefan Petry
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
Aventis Pharma Deutschland GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from DE2002100173 external-priority patent/DE10200173A1/en
Priority claimed from DE2002136329 external-priority patent/DE10236329A1/en
Application filed by Aventis Pharma Deutschland GmbH filed Critical Aventis Pharma Deutschland GmbH
Publication of EP1466009A2 publication Critical patent/EP1466009A2/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • 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
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • PTPase protein tyrosine phosphatase
  • the invention relates to an improved method for the detection of protein tyrosine phosphatases, in particular under neutral conditions, using 6,8-difluoro-4-methylumbelliferyl phosphate.
  • p-NPP substrate p-nitrophenyl phosphate
  • Such detection methods can be found in standard biochemistry manuals such as "Current protocols in protein science: John E. Coligan (ed.), Ben M. Dünn (ed.), Hidde L. Ploegh (ed.), David W. Speicher ( ed.), Paul T. Wingfield (ed.); SBN: 0-471-11184-8; loose-leaf work, continuously renewed; publisher John Wiley & Sons.' ⁇ us the p-NPP arises from the action of a phosphatase p-nitrophenol.
  • P-Nitrophenol can be detected photometrically due to its intense yellow color in the alkaline range
  • test procedure has some unfavorable properties.
  • the test is not suitable for the direct determination of enzyme activity since it is carried out according to the "time-stop" principle.
  • the enzyme reaction is interrupted after a certain period of time by adding sodium hydroxide solution.
  • the increase in pH leads to a color change of the resulting p-nitrophenol to yellow, the absorption of which is determined photometrically and is a measure of the amount of p-nitrophenol present.
  • This test principle is quite cumbersome for enzyme kinetic studies, for example to determine the type of inhibitor. Since a large number of measuring points are required, a correspondingly large number of coordinated individual approaches must be set up.
  • the substrate is also sensitive to light, temperature and pH. In the range of the physiological pH, it tends to decompose slowly. Some of the tyrosine phosphatases to be investigated have the maximum activity with pNPP as a substrate in the acidic range. PTP1 B, for example, shows the higher conversion at a pH value of 5.6. At the physiological pH value of 7.0, on the other hand, PTP1 B only works with pNPP as substrate with 30% of the maximum sales value. As a result, relatively high amounts of the enzyme are required, which makes the costs correspondingly more expensive. Another negative factor of this test method is that sometimes other components present in the test such as buffers, salts or other (test substances) have an absorption in the yellow range. Appropriate background checks are required for this.
  • the malchite green phosphopeptide test is described as a further proof of the activity of protein tyrosine phosphatases (Martin et al. (1985) Journal of Biological Chemistry, 260, pp. 14932 and Härder et al. (1994) Biochemical Journal, 298, pp. 395).
  • the inorganic phosphate released from the substrate peptide by the phosphatase is detected photometrically by the malachite green reagent.
  • a further disadvantage is that the specific substrate peptide must be provided in high concentration for each phosphatase, which generally makes the process quite expensive.
  • 3,6-Fluorescein diphosphate is described as another substrate for the detection of protein tyrosine phosphatases (Journal of Biomolecular Screening 4, 327-334, 1999). In contrast to the two substrates mentioned above, it enables direct measurement of enzyme activity, but the spectral ones are
  • the object of the present invention is therefore a further.
  • the invention relates to a method for the detection of the enzymatic activity of a protein tyrosine phosphatase in biological material, wherein a] biological material or a preparation made of biological material is provided,
  • the preparation from the biological material preferably consists of a protein tyrosine phosphatase from the group LAR, CD 45, PTP alpha, PTP 1 B, TCPTP, YOP, CDC 25, PTEN, SHP1, 2.
  • the preparation of the biological material can be in different purity levels.
  • the preparation from the biological material can be whole cells, disintegrations of cells, samples enriched with cell components and / or organelles, or purified proteins.
  • the concentration of the 6,8-difluoro-4-methylumbiferiferyl phosphate is preferably 10 to 250 ⁇ M when it is brought into contact with the biological material or the preparation from biological material.
  • the concentration of the DiFMUP is particularly preferably 50 to 100 ⁇ M.
  • the pH of the aqueous solution in which the biological material or the preparation of biological material is brought into contact with the DiFMUP is preferably between 5.0 and 8.0 and particularly preferably between 6.0 and 7.5. In another particularly preferred embodiment, this pH is 7.0.
  • the invention also relates to a method for identifying a compound which modifies the activity of a protein tyrosine phosphatase, wherein
  • a] a chemical compound is provided, b] biological material or a preparation of biological material is provided, c] 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) is provided, d] the chemical compound from a], as well as the biological Material or the preparation of biological material from b] and the DiFMUP from c] are brought into contact with one another in an aqueous solution, e] the amount of the 6,8-difluoro-4-methylumbelliferyl then formed is determined fluorometrically f, the amount of the resulting 6,8-difluoro-4-methylumbelliferyl from e] is compared with the amount of the resulting 6,8-difluoro-4-methylumbelliferyl in a control batch.
  • DiFMUP 6,8-difluoro-4-methylumbelliferyl phosphate
  • a control approach is characterized in particular by the fact that when the biological material or the preparation made of biological material is brought into contact with DiFMUP, either no chemical compound in the sense of the aforementioned process step a) is involved or the effect of the chemical compound in relation to a protein tyrosine Phosphatase of the chosen
  • Embodiment is already known.
  • Such chemical compounds which are used in the control batch and whose effect on a protein tyrosine phosphatase is already known, can in particular be vanadate, vanadium organic compounds, pervanadate, okadaic acid, NaF, dephostasin, modified peptides or other compounds.
  • this modification should consist of stimulating, inhibiting or stabilizing the activity of a protein tyrosine phosphatase.
  • this protein tyrosine phosphatase is selected from the group comprising LAR, CD 45, PTP alpha, TC-PTP, CDC 25, PTEN.YOP, SHP1.2., PTP1 B.
  • the invention also relates to a compound which modifies the activity of a protein tyrosine phosphatase and which has been identified by the above-described method for identifying a compound which modifies the activity of a protein tyrosine phosphatase.
  • This compound preferably has a mass between 0.1 to 50 kDa, further preferably between 0.1 to 5 kDa and further preferably between 0.1 to 3 kDa.
  • the compound can be a protein, an amino acid, a polynucleotide, a nucleotide, a natural product or an aromatic hydrocarbon compound.
  • the invention also relates to a pharmaceutical composition which comprises at least one compound as described above, which has been identified by a method for identifying a compound which modifies the activity of a protein tyrosine phosphatase.
  • the medicament further comprises formulation auxiliaries for a medicament and / or polymeric additives.
  • the invention also relates to the use of a compound identified by a method of identifying a compound that modifies the activity of a protein tyrosine phosphatase for the manufacture of a medicament for the treatment of diabetes.
  • 6,8-difluoro-4-methylumbelliferyl phosphatase is commercially available.
  • Molecular Probes Europe BV (2333 Leiden, The Netherlands) sells this chemical. The manufacture is disclosed in US 5,830,912.
  • Biomaterial is any material that contains genetic information and also bacteria or fungi such as Escherichia coli or Saccharomyces cerevisiae. Biological material also includes cells from cell cultures. In the case of cells from animal or human tissues, biological material can be obtained by biopsy, surgical removal, removal by means of syringes or catheters or comparable techniques. The cells removed in this way can be frozen, processed or taken in cell culture. Bacteria and yeast cells are propagated and processed using common microbiology techniques.
  • Biological material can also consist of the cells of an animal cell culture. Such cells are, for example, mouse cells, rat cells or hamster cells.
  • the cell culture cells can be primary cell types or established cell lines. Examples of established cell lines are mouse 3T3 cells, CHO cells or Heia cells. The keeping, cultivation and multiplication of cell lines is described in standard textbooks, for example in "Basic Cell Culture; ed .: JM Daris IRL Press, Oxford (1996)".
  • a preparation of a biological material is produced, for example, by digestion of the biological material and subsequent cleaning steps. Methods for disintegrating the biological material can in particular be repeated freezing and thawing, treatment with ultrasound, the use of a French press, the addition of detergents and enzymes or the like.
  • Subsequent cleaning steps consist, for example, of differential centrifugation, precipitation with ammonium sulfate or organic solvents, the use of chromatographic techniques and others.
  • Chromatographic techniques include polyacrylamide gel electrophoresis, high pressure liquid chromatography, ion exchange chromatography, affinity chromatography, gas chromatography, mass spectrometry and others.
  • Textbooks are available to the person skilled in the art for this and in particular also for detailed instructions on the pure presentation of proteins, such as in particular "Current Protocols in Protein Science, ed .: JE Coligan et al., LoseblattwerK, continuously renewed, edition 2001, published by John Wiley &Sons".
  • the biological material or the preparation made of biological material can be brought into contact with DiFMUP in conventional laboratory vessels such as Eppendorf tubes, centrifuge tubes or glass flasks.
  • the underlying aqueous medium contains, for example, buffer substances, nutrient medium components, monovalent or divalent ions such as Na + , K + , Ca 2+ , CI “ , SO 2" , PO 3 2 " or others, as well as proteins, glycerol or others.
  • aqueous solvent can in particular also contain a certain proportion of an organic solvent such as dimethyl sulfoxide, methanol or ethanol, but the content of such a solvent is preferably not more than 10% by volume of the batch.
  • the PTPase (phosphatase) protein family currently includes about 100 different members. These can be roughly divided into receptor-coupled and cytoplasmic.
  • the phosphatases have in common the amino acid motif (H / V) CX 5 R (S / T) in the catalytic domain.
  • the receptor-linked phosphatases usually consist of an extracellular domain, a single transmembrane region and one or two cytoplasmic PTPase domains.
  • the LAR (leukocyte common antigen-related) and the PTP ⁇ are included in the receptor-coupled PTPases.
  • the intracellular PTPases normally contain a catalytic domain and various extensions of the C- or N-terminal region, for example by "SH domains".
  • the enzyme PTP1 B is assigned to the cytoplasmic PTPases.
  • the PTPase family also includes the group of dual phosphatases. These enzymes are used alongside
  • Phosphotyrosine also phosphoserine or phosphothreonine as a substrate.
  • This group includes, for example, the phosphatases VHR and cdc25.
  • the phosphatases LAR, PTP, SHP-2 and PTP1 B are believed to have important functions in the insulin-mediated signaling pathway. These PTPases associate with the insulin receptor and catalyze dephosphorylation. These PTPases, individually or in combination, may play a role in the pathogenesis of insulin resistance. (Biochemistry 38, 3793-3803, 1999; p. 3799).
  • PTP1 B is a negative regulator of the signal transduction pathway stimulated by insulin, which means that the protein switches off the signal induced by insulin.
  • the signal pathway is probably interrupted by direct dephosphorylation of the insulin receptor.
  • PTP1 B is also overexpressed in a large percentage of breast cancer patients.
  • the enzyme interacts with the "epidermal growth factor”. Two aryl phosphate binding pockets were detected for the enzyme. One is located directly in the active center, another at a location adjacent to the catalytic center outside of this. (Biochemistry 38, 3793-3803, 1999).
  • the state of phosphorylation specifies a precisely balanced activity of complementary protein tyrosine kinases (PTK) and phosphatases (PTPase), in particular protein tyrosine phosphatases.
  • PTK complementary protein tyrosine kinases
  • PBPase phosphatases
  • PTPases are responsible for the selective dephosphorylation of phospho-tyrosine residues. PTPases work in conjunction with the protein tyrosine kinases in a variety of different biological processes when transmitting signals through, for example, growth factors or hormones. These signal transduction mechanisms play an important role in the control of cellular metabolism, growth, differentiation or mobility. The incorrect regulation of signaling pathways is discussed for some pathological processes as one of the causal conditions. These include cancer, some immunological and neurological diseases as well as type II diabetes and obesity.
  • a chemical compound is provided, for example, by chemical synthesis.
  • the standard synthetic methods are familiar to the person skilled in the art.
  • the chemical compound can be part of a collection of chemical compounds as they result from the storage and cataloging of chemical compounds from completed synthesis programs (so-called "chemical libraries").
  • the compound may have been formed by a microorganism, in particular a bacterium, but also by a fungus or a plant (natural product).
  • Suitable pharmaceutical compounds for oral administration can be in separate units, such as capsules, capsules, lozenges or tablets, as powders or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water - or water-in-oil emulsion.
  • These compositions can be prepared by any suitable pharmaceutical method comprising a step of contacting the active ingredient and the carrier (which may consist of one or more additional ingredients).
  • the compositions are prepared by uniformly and homogeneously mixing the active ingredient with a liquid and / or finely divided solid carrier, after which the product is shaped if necessary.
  • a tablet can be made by pressing or molding a powder or granules of a compound, optionally with one or more additional ingredients.
  • Compressed tablets can be prepared by tabletting the compound in free flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent and / or a (several) surface active / dispersing agent in a suitable machine.
  • Molded tablets can be made by molding the powdered compound moistened with an inert liquid diluent in a suitable machine.
  • compositions suitable for oral (sublingual) administration include lozenges that contain a compound with a flavor, usually sucrose and gum arabic or tragacanth, and lozenges that contain a compound in an inert base such as gelatin and glycerin or sucrose and gum include arabic.
  • compositions suitable for parenteral administration preferably comprise sterile aqueous preparations of a compound which are preferably isotonic with the blood of the intended recipient. These preparations are preferably administered intravenously, although they can also be administered subcutaneously, intramuscularly or intradermally as an injection. These preparations can preferably be prepared by mixing the compound with water and making the solution obtained sterile and isotonic with the blood. Injectable compositions according to the invention generally contain from 0.1 to 5% by weight of an active compound.
  • Suitable pharmaceutical compositions for rectal administration are preferably in the form of single-dose suppositories.
  • Suitable pharmaceutical compositions for topical use on the skin are preferably in the form of an ointment, cream, lotion, paste, spray, aerosol or oil.
  • Vaseline, lanolin, polyethylene glycols, alcohols and combinations of two or more of these substances can be used as carriers.
  • the active ingredient is generally present in a concentration of 0.1 to 15% by weight of the composition, for example 0.5 to 2%.
  • Suitable pharmaceutical compositions for transdermal applications can be presented as individual patches which are suitable for long-term close contact with the patient's epidermis.
  • Such plasters suitably contain the active ingredient in an optionally buffered aqueous solution, dissolved and / or dispersed in an adhesive or dispersed in a polymer.
  • a suitable active ingredient concentration is approximately 1% to 35%, preferably approximately 3% to 15%.
  • Type 2 diabetes is characterized by high glucose levels (hyperglycemia) in the fasted state (> 126mg / dl), insulin resistance in peripheral tissues such as muscle or fat, an increased gluconeogenesis of the liver, as well as insufficient insulin secretion the pancreatic ß cells. The actual cause of this disease is not yet known.
  • Type 2 diabetes is very common with other conditions such as obesity (obesity), hypertriglyceridaemia (high blood lipids) and high blood pressure.
  • Insulin resistance is believed to be a key to understanding the clinical picture. Insulin resistance manifests itself in the reduced extent of the peripheral organs reacting to a defined concentration of insulin. This is reflected at the cellular level, in the increase in the amount of insulin required to trigger an effect from insulin. Insulin produces various effects on glucose and fat metabolism in cells of muscle, fat and liver, such as increasing glucose uptake from the blood, increasing the glucose metabolism rate or inhibiting fatty acid cleavage. The emergence of insulin resistance at the cellular level is generally associated with various factors. The insulin receptor, the factors of the signal cascade and the components of the glucose transport system play an important role in this. In the first step, insulin effects its biological functions by binding to the insulin receptor.
  • insulin receptor kinase After binding of the insulin, this is subject to an autophosphorylation of the ⁇ -subunit by the insulin receptor kinase.
  • the signal is passed on in cells in muscle cells via IRS (insulin receptor substrate) and PI3K (phospho-inositol-3-kinase) and leads to the stimulation of glucose uptake.
  • Insulin has a number of other effects that take place via only partially known mechanisms.
  • specific kinases and phosphatases interact in a coordinated manner. The dissociation of the insulin from the receptor is not sufficient to switch off the signal induced by insulin.
  • the tyrosine kinase activity of the insulin receptor lasts as long as the regulatory domain remains phosphorylated. Shutdown takes place via cellular PTPases.
  • Pharmacologically active compounds with an inhibitory effect on negative regulators of the insulin signal path have this
  • reaction buffer contains the following components: protein tyrosine phosphatase PTP1 B in the desired final concentration ( Figure 1: 30 - 600 ng / ml); 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT.
  • the phosphatase reaction is started by adding 15 ⁇ l of 1 mM DiFMUP solution and the increase in fluorescence (measured in RFU) is measured continuously in a fluorescence microtiter plate photometer at 358 nm excitation and 455 nm emission wavelength over 15 minutes.
  • the measure of the enzyme activity is the increase in fluorescence as a function of the final PTP1B concentration, which can be shown graphically (FIG. 1).
  • reaction buffer contains the following components: 100 ng / ml protein tyrosine phosphatase PTPI b; 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT.
  • the phosphatase reaction is started by adding 15 ⁇ l DiFMUP solution, which contains the substrate in 10 times the concentration desired in the test mixture (Figure 2: 0 - 200 ⁇ M), and the fluorescence in a fluorescence microtiter plate photometer at 358/455 nm measured at intervals of 30 seconds over 15 minutes. Measure of the following components: 100 ng / ml protein tyrosine phosphatase PTPI b; 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT.
  • the phosphatase reaction is started by adding 15 ⁇ l DiFMUP solution, which contains the substrate in 10 times the concentration desired in the test mixture ( Figure 2:
  • Enzyme activity is the increase in fluorescence (measured in RFU) as a function of the DiFMUP concentration, which can be shown graphically (FIG. 2). From this, the kinetic constants of the enzyme reaction can then be determined using Lineweaver-Burk analysis. For PTP1 B this results in a Km value of 19 ⁇ M and a Vmax of 388000 RFU sec "1 mg " 1 . This analysis can also be carried out analogously for other tyrosine phosphatases. The kinetic constants can be found in Table 1. 3. Cleavage of DiFMUP depending on the enzyme concentration of the phosphotyrosine phosphatases PTPalpha, LAR, T cell-PTP, SHP-2, CD45 and YOP.
  • reaction buffer contains the following components: protein tyrosine phosphatase in the desired final concentration (FIG.
  • PTPalpha 0.5-1.85 ⁇ g / ml
  • LAR 125-500 ng / ml
  • Tcell-PTP 66 - 330 ng / ml
  • CD 45 50 - 400 ng / ml
  • YOP 50-400 ng / ml
  • SHP-2 0.3 - 2.4 ⁇ g / ml
  • the phosphatase reaction is started by adding 15 ⁇ l of 1 mM DiFMUP solution and the fluorescence is measured in a fluorescence microtiter plate photometer at 358/455 nm in time intervals of 30 seconds over 15 minutes.
  • the measure of the enzyme activity is the increase in fluorescence (measured in RFU) as a function of the final concentration of the protein tyrosine phosphatases, which can be shown graphically (FIG. 3).
  • the test for determining the inhibitory action of the active substance 2,2-dioxo-2,3-dihydro-2,6-benzo [1, 2,3] oxathiazol-5-yl) - (9-ethyl-9H-carbazol-3- ylmethyl) -amine using the DiFMUP test is carried out in a black microtiter plate at a temperature of 37 ° C.
  • 120 ⁇ l of reaction buffer are provided, which contains the following components: 100 ng / ml protein tyrosine phosphatase PTP1 B; 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT.
  • reaction buffer contains the following components: 100 ng / ml protein tyrosine phosphatase PTP1 B; 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT.
  • 15 ⁇ l of the inhibitor solution to be
  • Phosphatase reaction is started by adding 15 ⁇ l of 1 mM DiFMUP solution, and the fluorescence (measured in RFU) is measured in a fluorescence microtiter plate photometer at 358/455 nm at time intervals of 30 seconds over 15 minutes.
  • the measure of the enzyme activity is the increase in fluorescence, which can be represented graphically (FIG. 1). Depending on the used
  • Inhibitor concentration results in a reduction in the enzymatic activity.
  • the inhibitor concentration at which the active ingredient 2,2-dioxo-2,3-dihydro-2,6-benzo [1, 2,3] oxathiazol-5-yl) - (9-ethyl-9H-carbazol-3-ylmethyl ) -amine the activity of PTP1 B reduced by half (IC-50) can be determined with 3.8 ⁇ M.
  • the IC-50 value was determined using the pNPP test method and the malchite green phosphopeptide test. This results in an IC50 of 5.1 ⁇ M for the pNPP test method and an IC50 of 3.9 ⁇ M for the malchite green phosphopeptide test method.
  • the corresponding inhibition curves are also shown in FIG. 4.
  • reaction buffer contains the following components: 100 ng / ml protein tyrosine phosphatase PTPI b; 50 mM Hepes pH 6.9; 150 mM NaCl; 1 mM EDTA; 2 mM DTT and an inhibitor concentration that depends on the previously determined IC50.
  • the phosphatase reaction is started by adding 15 ⁇ l of DiFMUP solution, which contains the substrate with 10 times the desired final concentration in the final volume ( Figure 2: 0 - 200 ⁇ M) and the fluorescence in a fluorescence microtiter plate photometer at 358-455 nm measured at time intervals of 30 seconds over 15 minutes until the reaction saturates. A 10-fold excess of the final concentration of substrate used previously is then added and the reaction is further monitored in a fluorescence microtiter plate photometer at 358-455 nm at time intervals of 30 seconds over 15 minutes. In the presence of irreversible inhibitors, the reaction cannot be started again; this is possible with a reversible type of inhibitor (FIG. 5). 6. Characterization of the inhibitor type of a phosphatase inhibitor on PTPI b by time-dependent incubation.
  • reaction takes place in a black microtiter plate at a temperature of '37 ° C. 120 ul reaction buffer is provided, the following
  • Component contains: 100 ng / ml protein tyrosine phosphatase PTPIb; 50 mM Hepes pH 6.9; 150 mM NaCl; 1 mM EDTA; 2 mM DTT and an inhibitor concentration that depends on the previously determined IC50.
  • Fig. 1 Cleavage of DiFMUP depending on the PTP1 B enzyme concentration.
  • Fig. 2 concentration dependence of the cleavage of DiFMUP by PTP1B.
  • Fig. 4 Determination of the inhibitory effect of a phosphatase inhibitor of PTP1B.
  • Fig.6 Characterization of the inhibitor type of a phosphatase inhibitor by time-dependent incubation

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Abstract

The invention relates to a method for detecting a protein-tyrosine-phosphatase in biological material by using 6,8-difluoro-4-methylumbelliferylphosphate (DiFMUP).

Description

Beschreibung description
Ein hochsensitiver und kontinuierlicher Protein Tyrosin-Phosphatase (PTPase) Test unter Verwendung von 6,8 Difluoro-4-methyl-umbelliferylphosphat .A highly sensitive and continuous protein tyrosine phosphatase (PTPase) test using 6,8 difluoro-4-methyl-umbelliferyl phosphate.
Die Erfindung betrifft ein verbessertes Verfahren zum Nachweis von Protein-Tyrosin- Phosphatasen insbesondere unter neutralen Bedingungen mittels 6,8-Difluoro-4- methylumbelliferylphosphat.The invention relates to an improved method for the detection of protein tyrosine phosphatases, in particular under neutral conditions, using 6,8-difluoro-4-methylumbelliferyl phosphate.
Im Stand der Technik offenbart sind Verfahren zum Nachweis der Aktivität einer Protein-Tyrosin-Phosphatase, wobei das Substrat p-Nitrophenylphosphat (p-NPP) verwendet wird. Solche Nachweisverfahren finden sich in Standardhandbüchern der Biochemie wie beispielsweise in "Current protocols in protein science: John E. Coligan (ed.), Ben M. Dünn (ed.), Hidde L. Ploegh (ed.), David W. Speicher (ed.), Paul T. Wingfield (ed.); SBN: 0-471-11184-8; Loseblattwerk, laufend erneuert; Verlag John Wiley & Sons.'Αus dem p-NPP entsteht durch die Einwirkung einer Phosphatase p- Nitrophenol. P-Nitrophenol ist wegen seiner intensiven Gelbfärbung im alkalischen Bereich photometrisch nachweisbarMethods for detecting the activity of a protein tyrosine phosphatase are disclosed in the prior art, the substrate p-nitrophenyl phosphate (p-NPP) being used. Such detection methods can be found in standard biochemistry manuals such as "Current protocols in protein science: John E. Coligan (ed.), Ben M. Dünn (ed.), Hidde L. Ploegh (ed.), David W. Speicher ( ed.), Paul T. Wingfield (ed.); SBN: 0-471-11184-8; loose-leaf work, continuously renewed; publisher John Wiley & Sons.'Αus the p-NPP arises from the action of a phosphatase p-nitrophenol. P-Nitrophenol can be detected photometrically due to its intense yellow color in the alkaline range
Dieses Testverfahren weist allerdings einige ungünstige Eigenschaften auf. Der Test eignet sich nicht zur direkten Bestimmung der Enzymaktivität, da er nach dem "Time- Stop"-Prinzip durchgeführt wird. Die Enzymreaktion wird hierbei nach Ablauf einer bestimmten Zeitdauer durch Zugabe von Natronlauge unterbrochen. Der pH-Wert- Anstieg führt zu einem Farbumschlag des entstandenen p-Nitrophenols ins Gelbe, dessen Absorption photometrisch bestimmt wird und ein Maß für die vorhandene Menge an p-Nitrophenol darstellt. Dieses Testprinzip ist für enzymkinetische Untersuchungen beispielsweise zur Bestimmung des Hemmtyps recht umständlich. Da eine Vielzahl von Meßpunkten erforderlich sind, müssen entsprechend viele miteinander koordinierte Einzelansätze aufgebaut werden.However, this test procedure has some unfavorable properties. The test is not suitable for the direct determination of enzyme activity since it is carried out according to the "time-stop" principle. The enzyme reaction is interrupted after a certain period of time by adding sodium hydroxide solution. The increase in pH leads to a color change of the resulting p-nitrophenol to yellow, the absorption of which is determined photometrically and is a measure of the amount of p-nitrophenol present. This test principle is quite cumbersome for enzyme kinetic studies, for example to determine the type of inhibitor. Since a large number of measuring points are required, a correspondingly large number of coordinated individual approaches must be set up.
Das Substrat ist außerdem licht-, temperatur- und pH empfindlich. Im Bereich des physiologischen pH-Wertes neigt es zu langsamer Zersetzung. Einige der zu untersuchenden Tyrosinphosphatasen haben das Aktivitätsmaximum mit pNPP als Substrat im sauren Bereich. PTP1 B beispielsweise zeigt bei einem pH- Wert von 5,6 den stärkeren Umsatz. Beim physiologischen pH-Wert von 7,0 hingegen arbeitet PTP1 B mit pNPP als Substrat nur noch mit 30 % des maximalen Umsatzwertes. Dadurch werden relativ hohe Mengen des Enzyms benötigt, wodurch sich die Kosten entsprechend verteuern. Ein weiterer negativer Faktor dieses Testverfahrens liegt darin, daß bisweilen andere im Test vorhandene Komponenten wie Puffer, Salze oder anderes (Testsubstanzen) eine Absorption im gelben Bereich aufweisen. Dafür sind entsprechende Hintergrundkontrollen erforderlich.The substrate is also sensitive to light, temperature and pH. In the range of the physiological pH, it tends to decompose slowly. Some of the tyrosine phosphatases to be investigated have the maximum activity with pNPP as a substrate in the acidic range. PTP1 B, for example, shows the higher conversion at a pH value of 5.6. At the physiological pH value of 7.0, on the other hand, PTP1 B only works with pNPP as substrate with 30% of the maximum sales value. As a result, relatively high amounts of the enzyme are required, which makes the costs correspondingly more expensive. Another negative factor of this test method is that sometimes other components present in the test such as buffers, salts or other (test substances) have an absorption in the yellow range. Appropriate background checks are required for this.
Als weiterer Nachweis der Aktivität von Protein-Tyrosin-Phosphatasen ist der Malchitgrün-Phosphopeptidtest beschrieben (Martin et al. (1985) Journal of Biological Chemistry, 260, pp. 14932 und Härder et al. (1994) Biochemical Journal, 298, pp. 395). Hierbei wird das durch die Phosphatase aus ihrem Substratpeptid freigesetzte anorganische Phosphat durch das Malachitgrün-Reagenz photometrisch nachgewiesen. Neben der Anfälligkeit der photometrischen Bestimmung durch beispielsweise Verunreinigungen oder pH-Empfindlichkeit und der Umständlichkeit des Timestop-Verfahrens, ist ein weiterer Nachteil, daß für jede Phosphatase das spezifische Substratpeptid in hoher Konzentration bereitgestellt werden muß, welches das Verfahren im allgemeinen recht kostspielig werden lässt.The malchite green phosphopeptide test is described as a further proof of the activity of protein tyrosine phosphatases (Martin et al. (1985) Journal of Biological Chemistry, 260, pp. 14932 and Härder et al. (1994) Biochemical Journal, 298, pp. 395). The inorganic phosphate released from the substrate peptide by the phosphatase is detected photometrically by the malachite green reagent. In addition to the susceptibility of photometric determination due to, for example, impurities or pH sensitivity and the complexity of the timestop process, a further disadvantage is that the specific substrate peptide must be provided in high concentration for each phosphatase, which generally makes the process quite expensive.
Als anderes Substrat zum Nachweis von Protein-Tyrosin-Phosphatasen ist 3,6- Fluorescein Diphosphate beschrieben (Journal of Biomolecular Screening 4, 327- 334, 1999). Es ermöglicht im Gegensatz zu den beiden oben genannten Substraten zwar die direkte Messung der Enzymaktivität, allerdings sind die spektralen3,6-Fluorescein diphosphate is described as another substrate for the detection of protein tyrosine phosphatases (Journal of Biomolecular Screening 4, 327-334, 1999). In contrast to the two substrates mentioned above, it enables direct measurement of enzyme activity, but the spectral ones are
Eigenschaften dieses Substrats für Messungen im Bereich des physiologischen pH- Wertes noch nicht besonders gut.Properties of this substrate are not yet particularly good for measurements in the range of the physiological pH.
Die Aufgabe der vorliegenden Erfindung besteht deshalb darin, ein weiteres Die Erfindung betrifft ein Verfahren zum Nachweis der enzymatischen Aktivität einer Protein-Tyrosin-Phosphatase in biologischem Material, wobei a] biologisches Material oder eine Präparation aus biologischem Material bereitgestellt wird,The object of the present invention is therefore a further. The invention relates to a method for the detection of the enzymatic activity of a protein tyrosine phosphatase in biological material, wherein a] biological material or a preparation made of biological material is provided,
b] 6,8-Difluoro-4-methylumbelliferylphosphat (DiFMUP) bereitgestellt wird,b] 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) is provided,
c] das biologische Material oder die Präparation aus biologischem Material aus a] mit dem DiFMUP aus b] in einer wäßrigen Lösung in Kontakt gebracht wird,c] the biological material or the preparation of biological material from a] is brought into contact with the DiFMUP from b] in an aqueous solution,
d] das dann entstehende 6,8-Difluoro-4-methylumbelliferyl fluorometrisch nachgewiesen wird.d] the resulting 6,8-difluoro-4-methylumbelliferyl is detected fluorometrically.
Die Präparation aus dem biologischen Material besteht bevorzugt aus einer Protein Tyrosin Phosphatase aus der Gruppe LAR, CD 45, PTP alpha, PTP 1 B, TCPTP, YOP, CDC 25, PTEN, SHP1 ,2. Die Präparation des biologischen Materials kann in unterschiedlichen Reinheitsstufen vorliegen. Es kann sich bei der Präparation aus dem biologischen Material um ganze Zellen, Aufschlüsse von Zellen, mit Zellbestandteilen und/oder Organellen angereicherter Proben oder um gereinigte Proteine handeln.The preparation from the biological material preferably consists of a protein tyrosine phosphatase from the group LAR, CD 45, PTP alpha, PTP 1 B, TCPTP, YOP, CDC 25, PTEN, SHP1, 2. The preparation of the biological material can be in different purity levels. The preparation from the biological material can be whole cells, disintegrations of cells, samples enriched with cell components and / or organelles, or purified proteins.
Die Konzentration des 6,8-Difluoro-4-methyIumbelliferylphosphats (DiFMUP) beträgt beim Inkontaktbringen mit dem biologischen Material oder der Präparation aus biologischem Material bevorzugt 10 bis 250 μM. Besonders bevorzugt beträgt die Konzentration des DiFMUP 50 bis 100 μM.The concentration of the 6,8-difluoro-4-methylumbiferiferyl phosphate (DiFMUP) is preferably 10 to 250 μM when it is brought into contact with the biological material or the preparation from biological material. The concentration of the DiFMUP is particularly preferably 50 to 100 μM.
Der pH-Wert der wäßrigen Lösung, in welchem das Inkontaktbringen des biologischen Materials oder der Präparation aus biologischem Material mit dem DiFMUP erfolgt, liegt bevorzugt zwischen 5,0 und 8,0 und besonders bevorzugt zwischen 6,0 und 7,5. In einer nochmals besonders bevorzugten Ausführungsform beträgt dieser pH-Wert 7,0. Die Erfindung betrifft auch ein Verfahren zur Identifizierung einer Verbindung, welche die Aktivität einer Protein-Tyrosin-Phosphatase modifiziert, wobeiThe pH of the aqueous solution in which the biological material or the preparation of biological material is brought into contact with the DiFMUP is preferably between 5.0 and 8.0 and particularly preferably between 6.0 and 7.5. In another particularly preferred embodiment, this pH is 7.0. The invention also relates to a method for identifying a compound which modifies the activity of a protein tyrosine phosphatase, wherein
a] eine chemische Verbindung bereitgestellt wird, b] biologisches Material oder eine Präparation aus biologischem Material bereitgestellt wird, c] 6,8-Difluoro-4-methylumbelliferylphosphat (DiFMUP) bereitgestellt wird, d] die chemische Verbindung aus a], sowie das biologische Material oder die Präparation aus biologischem Material aus b] und das DiFMUP aus c] miteinander in einer wäßrigen Lösung in Kontakt gebracht werden, e] die Menge des dann entstehenden 6,8-Difluoro-4-methylumbelliferyls f fluorometrisch bestimmt wird, fj die Menge des entstehenden 6,8-Difluoro-4-methylumbelliferyls aus e] verglichen wird mit der Menge des entstehenden 6,8-Difluoro-4- methylumbelliferyls in einem Kontrollansatz.a] a chemical compound is provided, b] biological material or a preparation of biological material is provided, c] 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) is provided, d] the chemical compound from a], as well as the biological Material or the preparation of biological material from b] and the DiFMUP from c] are brought into contact with one another in an aqueous solution, e] the amount of the 6,8-difluoro-4-methylumbelliferyl then formed is determined fluorometrically f, the amount of the resulting 6,8-difluoro-4-methylumbelliferyl from e] is compared with the amount of the resulting 6,8-difluoro-4-methylumbelliferyl in a control batch.
Ein Kontrollansatz zeichnet sich insbesondere dadurch aus, daß beim Inkontaktbringen des biologischen Materials oder der Präparation aus biologischem Material mit DiFMUP entweder keine chemische Verbindung im Sinne des zuvor genannten Verfahrensschritts a) beteiligt ist oder die Wirkung der chemischen Verbindung in Bezug auf eine Protein-Tyrosin-Phosphatase der gewähltenA control approach is characterized in particular by the fact that when the biological material or the preparation made of biological material is brought into contact with DiFMUP, either no chemical compound in the sense of the aforementioned process step a) is involved or the effect of the chemical compound in relation to a protein tyrosine Phosphatase of the chosen
Ausführungsform bereits bekannt ist. Solche chemischen Verbindungen, welche im Kontrollansatz verwendet werden und deren Wirkung auf eine Protein-Tyrosin- Phosphatase bereits bekannt ist, können insbesondere Vanadat, Vanadium organische Verbindungen, Pervanadat, Okadaic-Säure, NaF, Dephostasin, modifizierte Peptide oder andere Verbindungen sein.Embodiment is already known. Such chemical compounds, which are used in the control batch and whose effect on a protein tyrosine phosphatase is already known, can in particular be vanadate, vanadium organic compounds, pervanadate, okadaic acid, NaF, dephostasin, modified peptides or other compounds.
In verschiedenen bevorzugten Ausführungsformen des Verfahrens zur Identifizierung einer Verbindung, welche die Aktivität einer Protein-Tyrosin-Phosphatase modifiziert, soll diese Modifizierung aus einer Stimulierung, Hemmung oder der Stabilisierung der Aktivität einer Protein-Tyrosin-Phosphatase bestehen. In einer weiteren bevorzugten Ausführungsform des Verfahrens zur Identifizierung einer Verbindung, welche die Aktivität einer Protein-Tyrosin-Phosphatase modifziert, wird diese Protein-Tyrosin-Phosphatase ausgewählt aus der Gruppe umfassend aus der Gruppe LAR, CD 45, PTP alpha, TC-PTP, CDC 25, PTEN.YOP, SHP1.2., PTP1 B.In various preferred embodiments of the method for identifying a compound which modifies the activity of a protein tyrosine phosphatase, this modification should consist of stimulating, inhibiting or stabilizing the activity of a protein tyrosine phosphatase. In a further preferred embodiment of the method for identifying a compound which modifies the activity of a protein tyrosine phosphatase, this protein tyrosine phosphatase is selected from the group comprising LAR, CD 45, PTP alpha, TC-PTP, CDC 25, PTEN.YOP, SHP1.2., PTP1 B.
Die Erfindung bezieht sich auch auf eine Verbindung, welche die Aktivität einer Protein- Tyrosin-Phosphatase modifiziert und welche durch vorstehend beschriebenes Verfahren zur Identifizierung einer Verbindung, welche die Aktivität einer Protein- Tyrosin-Phosphatase modifiziert, identifiziert wurde. Diese Verbindung hat bevorzugt eine Masse zwischen 0,1 bis 50 kDa, weiterhin bevorzugt zwischen 0,1 bis 5 kDa und weiterhin bevorzugt zwischen 0,1 bis 3 kDa. Die Verbindung kann ein Protein, eine Aminosäure, ein Polynucleotid, ein Nucleotid, ein Naturstoff oder eine aromatische Kohlenwasserstoffverbindung sein. Die Erfindung betrifft auch ein Arzneimittel, welches mindestens eine wie vorstehend beschriebene Verbindung umfaßt, die durch ein Verfahren zur Identifizierung einer Verbindung, welche die Aktivität einer Protein- Tyrosin-Phosphatase modifiziert, identifiziert wurde. Das Arzneimittel umfaßt weiterhin Formulierungshilfsstoffe für ein Arzneimittel und/oder polymere Zusatzstoffe.,The invention also relates to a compound which modifies the activity of a protein tyrosine phosphatase and which has been identified by the above-described method for identifying a compound which modifies the activity of a protein tyrosine phosphatase. This compound preferably has a mass between 0.1 to 50 kDa, further preferably between 0.1 to 5 kDa and further preferably between 0.1 to 3 kDa. The compound can be a protein, an amino acid, a polynucleotide, a nucleotide, a natural product or an aromatic hydrocarbon compound. The invention also relates to a pharmaceutical composition which comprises at least one compound as described above, which has been identified by a method for identifying a compound which modifies the activity of a protein tyrosine phosphatase. The medicament further comprises formulation auxiliaries for a medicament and / or polymeric additives.
Die Erfindung betrifft auch die Verwendung einer Verbindung, welche durch ein Verfahren zur Identifizierung einer Verbindung, welche die Aktivität einer Protein- Tyrosin-Phosphatase modifiziert, identifiziert wurde, zur Herstellung eines Arzneimittels zur Behandlung von Diabetes.The invention also relates to the use of a compound identified by a method of identifying a compound that modifies the activity of a protein tyrosine phosphatase for the manufacture of a medicament for the treatment of diabetes.
6,8-Difluoro-4-methylumbelliferyl-phosphatase ist kommerziell erhältlich. Beispielsweise vertreibt die Firma Molecular Probes Europe BV (2333 Leiden, The Netherlands) diese Chemikalie. Die Herstellung ist in US 5,830, 912 offenbart.6,8-difluoro-4-methylumbelliferyl phosphatase is commercially available. For example, Molecular Probes Europe BV (2333 Leiden, The Netherlands) sells this chemical. The manufacture is disclosed in US 5,830,912.
Biologisches Material ist jedes Material, das genetische Informationen enthält und sich auch Bakterien oder Pilze wie beispielsweise Escherichia coli oder Saccharomyces cerevisiae. Weiterhin umfaßt biologisches Material Zellen aus Zellkulturen. Die Gewinnung von biologischem Material kann im Falle von Zellen aus tierischen oder menschlichen Geweben durch Biopsie, operative Entnahme, Entnahme mittels Spritzen oder Kathetern oder vergleichbaren Techniken erfolgen. Die so entnommenen Zellen können tiefgefroren, aufgearbeitet oder in Zellkultur genommen werden. Bakterien und Hefezellen werden mittels gebräuchlicher Techniken der Mikrobiologie vermehrt und aufgearbeitet.Biological material is any material that contains genetic information and also bacteria or fungi such as Escherichia coli or Saccharomyces cerevisiae. Biological material also includes cells from cell cultures. In the case of cells from animal or human tissues, biological material can be obtained by biopsy, surgical removal, removal by means of syringes or catheters or comparable techniques. The cells removed in this way can be frozen, processed or taken in cell culture. Bacteria and yeast cells are propagated and processed using common microbiology techniques.
Entsprechende Anleitungen hierfür findet der Fachmann in "Current Protocols in Molecular Biology; ed.: F.M. Ansabel et al., Loseblattwerk, laufend erneuert, Ausgabe 2001 , Verlag John Wiley & Sons".Appropriate instructions for this can be found in "Current Protocols in Molecular Biology; ed .: F.M. Ansabel et al., Loseblattwerk, continuously renewed, edition 2001, publisher John Wiley & Sons".
Biologisches Material kann auch aus den Zellen einer tierischen Zellkultur bestehen. Solche Zellen sind beispielsweise Mauszellen, Rattenzellen oder Hamsterzellen. Die Zellkulturzellen können primäre Zelltypen oder etablierte Zelllinien sein. Beispiele für etablierte Zelllinien sind Maus 3T3-Zellen, CHO-Zellen oder Heia-Zellen. Haltung, Anzucht und Vermehrung von Zelllinien ist in Standardlehrbüchern beschrieben, wie beispielsweise in "Basic Cell Culture; ed.: J.M. Daris IRL Press, Oxford (1996)". Eine Präparation eines biologischen Materials wird beispielsweise durch Aufschluß des biologischen Materials und sich daran anschließende Reinigungsschritte hergestellt. Methoden zum Aufschluß des biologischen Materials können insbesondere wiederholtes Einfrieren und Auftauen, die Behandlung mit Ultraschall, die Verwendung einer French-Press, die Zugabe von Detergentien und Enzymen oder vergleichbares sein. Sich anschließende Reinigungsschritte bestehen beispielsweise aus differentieller Zentrifugation, der Fällung mit Ammonsulfat oder organischen Lösungsmitteln, der Anwendung von chromatographischen Techniken und anderen. Chromatographische Techniken sind beispielsweise die Polyacrylamidgelektrophorese, Hochdruckflüssigkeits-chromatographie, lonenaustauschchromatographie, Affinitätschromatographie, Gaschromatographie, Massenspektrometrie und anderes sein. Hierfür und insbesondere auch für detaillierte Anweisungen zur Reindarstellung von Proteinen stehen dem Fachmann Lehrbücher zur Verfügung wie insbesondere "Current Protocols in Protein Science, ed.: J. E. Coligan et al., LoseblattwerK, laufend erneuert, Ausgabe 2001 , Verlag John Wiley & Sons". Das Inkontaktbringen des biologischen Materials oder der Präparation aus biologischem Material mit DiFMUP kann in gewöhnlichen Laborgefäßen wie beispielsweise Eppendorf-Gefäßen, Zentrifugen-Röhrchen oder Glaskolben erfolgen. Das zugrundeliegende wäßrige Medium enthält beispielsweise Puffersubstanzen, Nährmediumbestandteile, einwertige oder zweiwertige Ionen wie Na+, K+, Ca2+, CI", SO 2", PO3 2" oder andere, weiterhin Proteine, Glycerin oder anderes. Für das Inkontaktbringen können bestimmte konstante Bedingungen wie insbesondere die Temperatur, der pH-Wert, die lonenbedingungen, die Konzentration eines Proteins, des Volumens oder andere Faktoren vorteilhaft sein. Dies wird erreicht, in dem beispielsweise das Inkontaktbringen in konstant temperierten Inkubationsvorrichtungen, in Gegenwart eines Puffers oder mit den zuvor genau eingewogenen Mengen der Ionen oder Proteine durchgeführt wird. Das wäßrige Lösungsmittel kann insbesondere auch einen bestimmten Anteil eines organischen Lösungsmittels wie Dimethylsulfoxid, Methanol oder Ethanol enthalten. Der Gehalt eines solchen Lösungsmittels beträgt aber vorzugsweise nicht mehr als 10 Vol. % des Ansatzes.Biological material can also consist of the cells of an animal cell culture. Such cells are, for example, mouse cells, rat cells or hamster cells. The cell culture cells can be primary cell types or established cell lines. Examples of established cell lines are mouse 3T3 cells, CHO cells or Heia cells. The keeping, cultivation and multiplication of cell lines is described in standard textbooks, for example in "Basic Cell Culture; ed .: JM Daris IRL Press, Oxford (1996)". A preparation of a biological material is produced, for example, by digestion of the biological material and subsequent cleaning steps. Methods for disintegrating the biological material can in particular be repeated freezing and thawing, treatment with ultrasound, the use of a French press, the addition of detergents and enzymes or the like. Subsequent cleaning steps consist, for example, of differential centrifugation, precipitation with ammonium sulfate or organic solvents, the use of chromatographic techniques and others. Chromatographic techniques include polyacrylamide gel electrophoresis, high pressure liquid chromatography, ion exchange chromatography, affinity chromatography, gas chromatography, mass spectrometry and others. Textbooks are available to the person skilled in the art for this and in particular also for detailed instructions on the pure presentation of proteins, such as in particular "Current Protocols in Protein Science, ed .: JE Coligan et al., LoseblattwerK, continuously renewed, edition 2001, published by John Wiley &Sons". The biological material or the preparation made of biological material can be brought into contact with DiFMUP in conventional laboratory vessels such as Eppendorf tubes, centrifuge tubes or glass flasks. The underlying aqueous medium contains, for example, buffer substances, nutrient medium components, monovalent or divalent ions such as Na + , K + , Ca 2+ , CI " , SO 2" , PO 3 2 " or others, as well as proteins, glycerol or others. Can be brought into contact certain constant conditions such as particularly temperature, pH, ionic conditions, concentration of a protein, volume or other factors can be advantageous, for example by contacting in constant temperature incubation devices, in the presence of a buffer or with the The aqueous solvent can in particular also contain a certain proportion of an organic solvent such as dimethyl sulfoxide, methanol or ethanol, but the content of such a solvent is preferably not more than 10% by volume of the batch.
Die PTPase (Phosphatase) Protein-Familie umfaßt gegenwärtig etwa 100 verschiedene Mitglieder. Diese können grob unterteilt werden in rezeptorgekoppelte und cytoplasmatische. Den Phosphatasen ist das Aminosäuremotiv (H/V)CX5R(S/T) in der katalytischen Domäne gemeinsam. Die rezeptorgekoppelten Phosphatasen bestehen gewöhnlich aus einer extrazellulären Domäne, einer einzigen Transmembranregion und einer oder zwei cytoplasmatischen PTPase Domänen. Zu den rezeptorgekoppelten PTPasen werden die LAR (leukocyte common antigen-related) und die PTPσ gerechnet. Die intrazellulären PTPasen enthalten normalerweise eine katalytische Domäne und verschiedene Verlängerungen des C- oder N-terminalen Bereichs beispielsweise durch "SH Domänen".The PTPase (phosphatase) protein family currently includes about 100 different members. These can be roughly divided into receptor-coupled and cytoplasmic. The phosphatases have in common the amino acid motif (H / V) CX 5 R (S / T) in the catalytic domain. The receptor-linked phosphatases usually consist of an extracellular domain, a single transmembrane region and one or two cytoplasmic PTPase domains. The LAR (leukocyte common antigen-related) and the PTPσ are included in the receptor-coupled PTPases. The intracellular PTPases normally contain a catalytic domain and various extensions of the C- or N-terminal region, for example by "SH domains".
Diesen Verlängerungen werden Funktionen bei der Zielfindung ("targeting") oder Regulation zugesprochen. Das Enzym PTP1 B wird den cytoplasmatischen PTPasen zugeordnet. Neben den reinen Tyrosin-Phosphatasen zählt zur PTPase Familie auch die Gruppe der dualen Phosphatasen. Diese Enzyme verwenden nebenFunctions in targeting or regulation are assigned to these extensions. The enzyme PTP1 B is assigned to the cytoplasmic PTPases. In addition to pure tyrosine phosphatases, the PTPase family also includes the group of dual phosphatases. These enzymes are used alongside
Phosphotyrosin auch Phosphoserin oder Phosphothreonin als Substrat. Zu dieser Gruppe gehören beispielsweise der Phosphatasen VHR und cdc25. Den Phosphatasen LAR, PTP , SHP-2 und PTP1 B werden wichtige Funktionen im Insulin vermittelten Signalweg zugesprochen. Diese PTPasen assoziieren mit dem Insulinrezeptor und katalysieren die Dephosphorylierung. Möglicherweise spielen diese PTPasen einzeln oder in Kombination eine Rolle bei der Pathogenese der Insulinresistenz. (Biochemistry 38, 3793-3803, 1999; S. 3799).Phosphotyrosine also phosphoserine or phosphothreonine as a substrate. This group includes, for example, the phosphatases VHR and cdc25. The phosphatases LAR, PTP, SHP-2 and PTP1 B are believed to have important functions in the insulin-mediated signaling pathway. These PTPases associate with the insulin receptor and catalyze dephosphorylation. These PTPases, individually or in combination, may play a role in the pathogenesis of insulin resistance. (Biochemistry 38, 3793-3803, 1999; p. 3799).
PTP1 B ist ein negativer Regulator des von Insulin stimulierten Signaltransduktionswegs, das heißt, das Protein schaltet das von Insulin induzierte Signal wieder ab. Vermutlich geschieht die Unterbrechung des Signalwegs durch direkte Dephosphorylierung des Insulinrezeptors. PTP1 B wird auch bei einem großen Prozentsatz von Patienten mit Brustkrebs überexprimiert. Darüberhinaus zeigt das Enzym Wechselwirkungen mit dem "epidermal growth factor". Für das Enzym konnten zwei Aryl-Phosphat-Bindetaschen nachgewiesen werden. Eine befindet sich direkt im aktiven Zentrum, eine weitere an einer dem katalytischen Zentrum benachbarten Stelle außerhalb von diesem. (Biochemistry 38, 3793-3803, 1999).PTP1 B is a negative regulator of the signal transduction pathway stimulated by insulin, which means that the protein switches off the signal induced by insulin. The signal pathway is probably interrupted by direct dephosphorylation of the insulin receptor. PTP1 B is also overexpressed in a large percentage of breast cancer patients. In addition, the enzyme interacts with the "epidermal growth factor". Two aryl phosphate binding pockets were detected for the enzyme. One is located directly in the active center, another at a location adjacent to the catalytic center outside of this. (Biochemistry 38, 3793-3803, 1999).
Die Weiterleitung und Beendigung von vielen intrazellulären Signalen wird durch Tyrosinphosphorylierung der beteiligten Faktoren gesteuert. DenThe forwarding and termination of many intracellular signals is controlled by tyrosine phosphorylation of the factors involved. The
Phosphorylierungszustand legt eine genau ausbalancierte Aktivität sich ergänzender Protein-Tyrosin-Kinasen (PTK) und Phosphatasen (PTPase) insbesondere Protein- Tyrosin-Phosphatasen fest.The state of phosphorylation specifies a precisely balanced activity of complementary protein tyrosine kinases (PTK) and phosphatases (PTPase), in particular protein tyrosine phosphatases.
PTPasen sind als Enzyme verantwortlich für die selektive Dephosphorylierung von Phospho-Tyrosin-Resten. PTPasen funktionieren im Zusammenspiel mit den Protein-Tyrosin-Kinasen vielfältig in unterschiedlichen biologischen Prozessen bei der Vermittlung von Signalen durch beispielsweise Wachstumsfaktoren oder Hormone. Diese Signaltransduktionsmechanismen spielen eine wichtige Rolle bei der Kontrolle des zellulären Stoffwechsels, des Wachstums, der Differenzierung oder Mobilität. Die fehlerhafte Regulierung von Signalwegen wird für einige pathologische Prozesse als eine der ursächlichen Bedingungen diskutiert. Dazu zählen beispielsweise Krebs, einige immunologische und neurologische Krankheiten sowie Diabetes vom Typ II und Obesitas.As enzymes, PTPases are responsible for the selective dephosphorylation of phospho-tyrosine residues. PTPases work in conjunction with the protein tyrosine kinases in a variety of different biological processes when transmitting signals through, for example, growth factors or hormones. These signal transduction mechanisms play an important role in the control of cellular metabolism, growth, differentiation or mobility. The incorrect regulation of signaling pathways is discussed for some pathological processes as one of the causal conditions. These include cancer, some immunological and neurological diseases as well as type II diabetes and obesity.
Die Bereitstellung einer chemischen Verbindung erfolgt beispielsweise durch chemische Synthese. Dem Fachmann sind die Standardsynthesemethoden geläufig. Die chemische Verbindung kann Teil einer Sammlung chemischer Verbindungen sein, wie sie durch Lagerung und Katalogisierung der chemischen Verbindungen aus abgeschlossenen Syntheseprogrammen entstehen (sogenannte "chemical libraries"). Die Verbindung kann in anderen Fällen von einem Mikroorganismus insbesondere einem Bakterium, aber auch von einem Pilz oder einer Pflanze gebildet worden sein (Naturstoff).A chemical compound is provided, for example, by chemical synthesis. The standard synthetic methods are familiar to the person skilled in the art. The chemical compound can be part of a collection of chemical compounds as they result from the storage and cataloging of chemical compounds from completed synthesis programs (so-called "chemical libraries"). In other cases, the compound may have been formed by a microorganism, in particular a bacterium, but also by a fungus or a plant (natural product).
Geeignete pharmazeutische Verbindungen für die orale Verabreichung können in separaten Einheiten vorliegen, wie zum Beispiel Kapseln, Oblatenkapseln, Lutschtabletten oder Tabletten, als Pulver oder Granulate, als Lösung oder Supension in einer wäßrigen oder nicht-wäßrigen Flüssigkeit, oder als eine Öl-in-Wasser- oder Wasser-in Öl-Emulsion. Diese Zusammensetzungen können nach jeder geeigneten pharmazeutischen Methode zubereitet werden, die einen Schritt umfaßt, bei dem der Wirkstoff und der Träger (der aus einem oder mehreren zusätzlichen Bestandteilen bestehen kann) in Kontakt gebracht werden. Im allgemeinen werden die Zusammensetzungen durch gleichmäßiges und homogenes Vermischen des Wirkstoffs mit einem flüssigen und/oder feinverteilten festen Träger hergestellt, wonach das Produkt, falls erforderlich, geformt wird.Suitable pharmaceutical compounds for oral administration can be in separate units, such as capsules, capsules, lozenges or tablets, as powders or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water - or water-in-oil emulsion. These compositions can be prepared by any suitable pharmaceutical method comprising a step of contacting the active ingredient and the carrier (which may consist of one or more additional ingredients). In general, the compositions are prepared by uniformly and homogeneously mixing the active ingredient with a liquid and / or finely divided solid carrier, after which the product is shaped if necessary.
So kann beispielsweise eine Tablette hergestellt werden, indem ein Pulver oder Granulat einer Verbindung verpreßt oder geformt wird, gegebenenfalls mit einem oder mehreren zusätzlichen Bestandteilen. Gepreßte Tabletten können durch Tablettieren der Verbindung in frei fließender Form, wie beispielsweise einem Pulver oder Granulat, gegebenenfalls gemischt mit einem Bindemittel, Gleitmittel, inertem Verdünner und/oder einem (mehreren) oberflächenaktiven/dispergierenden Mittel in einer geeigneten Maschine hergestellt werden. Geformte Tabletten können durch Formen der pulverförmigen, mit einem inerten flüssigen Verdünnungsmittel befeuchteten Verbindung in einer geeigneten Maschine hergestellt werden.For example, a tablet can be made by pressing or molding a powder or granules of a compound, optionally with one or more additional ingredients. Compressed tablets can be prepared by tabletting the compound in free flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent and / or a (several) surface active / dispersing agent in a suitable machine. Molded tablets can be made by molding the powdered compound moistened with an inert liquid diluent in a suitable machine.
Pharmazeutische Zusammensetzungen, die für eine perorale (sublinguale) Verabreichung geeignet sind, umfassen Lutschtabletten, die eine Verbindung mit einem Geschmacksstoff enthalten, üblicherweise Saccharose und Gummi arabicum oder Tragant, und Pastillen die eine Verbindung in einer inerten Basis wie Gelatine und Glycerin oder Saccharose und Gummi arabicum umfassen.Pharmaceutical compositions suitable for oral (sublingual) administration include lozenges that contain a compound with a flavor, usually sucrose and gum arabic or tragacanth, and lozenges that contain a compound in an inert base such as gelatin and glycerin or sucrose and gum include arabic.
Geeignete pharmazeutische Zusammensetzungen für die parenterale Verabreichung umfassen vorzugsweise sterile wäßrige Zubereitungen einer Verbindung, die vorzugsweise isotonisch mit dem Blut des vorgesehenen Empfängers sind. Diese Zubereitungen werden vorzugsweise intravenös verabreicht, wenngleich die Verabreichung auch subkutan, intramuskulär oder intradermal als Injektion erfolgen kann. Diese Zubereitungen können vorzugsweise hergestellt werden, indem die Verbindung mit Wasser gemischt wird und die erhaltene Lösung steril und mit dem Blut isotonisch gemacht wird. Injizierbare erfindungsgemäße Zusammensetzungen enthalten im allgemeinen von 0,.1 bis 5 Gew.-% einer aktiven Verbindung.Pharmaceutical compositions suitable for parenteral administration preferably comprise sterile aqueous preparations of a compound which are preferably isotonic with the blood of the intended recipient. These preparations are preferably administered intravenously, although they can also be administered subcutaneously, intramuscularly or intradermally as an injection. These preparations can preferably be prepared by mixing the compound with water and making the solution obtained sterile and isotonic with the blood. Injectable compositions according to the invention generally contain from 0.1 to 5% by weight of an active compound.
Geeignete pharmazeutische Zusammensetzungen für die rektale Verabreichung liegen vorzugsweise als Einzeldosis-Zäpfchen vor.Suitable pharmaceutical compositions for rectal administration are preferably in the form of single-dose suppositories.
Diese können hergestellt werden, indem man eine Verbindung mit einem oder mehreren herkömmlichen festen Trägern, beispielsweise Kakaobutter, mischt und das entstehende Gemisch in Form bringt.These can be made by mixing a compound with one or more conventional solid carriers, e.g. cocoa butter, and shaping the resulting mixture.
Geeignete pharmazeutische Zusammensetzungen für die topische Anwendung auf der Haut liegen vorzugsweise als Salbe, Creme, Lotion, Paste, Spray, Aerosol oder Öl vor. Als Träger können Vaseline, Lanolin, Polyethylenglykole, Alkohole und Kombinationen von zwei oder mehreren dieser Substanzen verwendet werden. Der Wirkstoff ist im allgemeinen in einer Konzentration von 0,1 bis 15 Gew.-% der Zusammensetzung vorhanden, beispielsweise von 0,5 bis 2 %.Suitable pharmaceutical compositions for topical use on the skin are preferably in the form of an ointment, cream, lotion, paste, spray, aerosol or oil. Vaseline, lanolin, polyethylene glycols, alcohols and combinations of two or more of these substances can be used as carriers. The active ingredient is generally present in a concentration of 0.1 to 15% by weight of the composition, for example 0.5 to 2%.
Auch eine transdermale Verabreichung ist möglich. Geeignete pharmazeutische Zusammensetzungen für transdermale Anwendungen können als einzelne Pflaster vorliegen, die für einen langzeitigen engen Kontakt mit der Epidermis des Patienten geeignet sind. Solche Pflaster enthalten geeigneterweise den Wirkstoff in einer gegebenenfalls gepufferten wäßrigen Lösung, gelöst und/oder dispergiert in einem Haftmittel oder dispergiert in einem Polymer. Eine geeignete Wirkstoff-Konzentration beträgt ca. 1 % bis 35 %, vorzugsweise ca. 3 % bis 15 %.Transdermal administration is also possible. Suitable pharmaceutical compositions for transdermal applications can be presented as individual patches which are suitable for long-term close contact with the patient's epidermis. Such plasters suitably contain the active ingredient in an optionally buffered aqueous solution, dissolved and / or dispersed in an adhesive or dispersed in a polymer. A suitable active ingredient concentration is approximately 1% to 35%, preferably approximately 3% to 15%.
Typ 2 Diabetes (NIDDM - non insulin dependent diabetes mellitus) ist charakterisiert durch hohe Glukosewerte (Hyperglycämie) im nüchternen Zustand (> 126mg/dl), Insulinresistenz in peripheren Geweben wie Muskel oder Fett, einer erhöhten Glukoneogenese der Leber, sowie einer ungenügenden Insulinsekretion durch die pankreatischen ß-Zellen. Die eigentliche Ursache dieser Erkrankung ist noch nicht bekannt. Der Typ 2 Diabetes tritt sehr häufig zusammen mit anderen Krankheitsbildern wie Obesitas (Fettleibigkeit), Hypertriglyceridämie (erhöhte Blutfettwerte) und Bluthochdruck auf.Type 2 diabetes (NIDDM - non insulin dependent diabetes mellitus) is characterized by high glucose levels (hyperglycemia) in the fasted state (> 126mg / dl), insulin resistance in peripheral tissues such as muscle or fat, an increased gluconeogenesis of the liver, as well as insufficient insulin secretion the pancreatic ß cells. The actual cause of this disease is not yet known. Type 2 diabetes is very common with other conditions such as obesity (obesity), hypertriglyceridaemia (high blood lipids) and high blood pressure.
Ein Schlüssel zum Verständnis des Krankheitsbildes wird in der Insulinresistenz vermutet. Die Insulinresistenz äußert sich im verringerten Ausmaß der peripheren Organe auf eine definierte Konzentration von Insulin zu reagieren. Dies spiegelt sich auf zellulärem Niveau wieder, in der Erhöhung der Insulinmenge, die benötigt wird, um einen Effekt durch Insulin auszulösen. Insulin erzeugt in Zellen von Muskel, Fett und Leber verschiedene Auswirkungen auf den Glukose- und Fettstoffwechsel wie Erhöhung der Glukoseaufnahme aus dem Blut, Erhöhung der Glukosestoffwechselrate oder Inhibierung der Fettsäurespaltung. Die Entstehung der Insulinresistenz auf zellulärem Niveau wird grundsätzlich mit verschiedenen Faktoren in Zusammenhang gebracht. Eine wichtige Rolle dabei spielen der Insulinrezeptor, die Faktoren der Signalkaskade und die Komponenten des Glukosetransportsystems. Insulin bewirkt seine biologischen Funktionen im ersten Schritt über eine Bindung an den Insulinrezeptor. Dieser unterliegt nach Bindung des Insulins einer Autophosphorylierung der ß-Untereinheit durch die Insulinrezeptorkinase. Das Signal wird in Muskelzellen zellulär weitervermittelt über IRS (Insulin Rezeptor Substrat) und PI3K (Phospho-lnositol-3-Kinase) und führt zur Stimulierung der Glukoseaufnahme. Insulin bewirkt eine Vielzahl anderer Effekte, die über nur teilweise bekannte Mechanismen ablaufen. Bei der intrazellulären Signalvermittlung agieren spezifische Kinasen und Phosphatasen in abgestimmter Weise zusammen. Für die Abschaltung des durch Insulin induzierten Signals reicht die Dissoziierung des Insulins vom Rezeptor nicht. Die Tyrosinkinaseaktivität des Insulinrezeptors hält so lange vor, als die regulatorische Domäne phosphoryliert bleibt. Die Abschaltung erfolgt über zelluläre PTPasen. Pharmakologisch wirksame Verbindungen mit Hemmwirkung auf negative Regulatoren des Insulinsignalweges haben dasInsulin resistance is believed to be a key to understanding the clinical picture. Insulin resistance manifests itself in the reduced extent of the peripheral organs reacting to a defined concentration of insulin. This is reflected at the cellular level, in the increase in the amount of insulin required to trigger an effect from insulin. Insulin produces various effects on glucose and fat metabolism in cells of muscle, fat and liver, such as increasing glucose uptake from the blood, increasing the glucose metabolism rate or inhibiting fatty acid cleavage. The emergence of insulin resistance at the cellular level is generally associated with various factors. The insulin receptor, the factors of the signal cascade and the components of the glucose transport system play an important role in this. In the first step, insulin effects its biological functions by binding to the insulin receptor. After binding of the insulin, this is subject to an autophosphorylation of the β-subunit by the insulin receptor kinase. The signal is passed on in cells in muscle cells via IRS (insulin receptor substrate) and PI3K (phospho-inositol-3-kinase) and leads to the stimulation of glucose uptake. Insulin has a number of other effects that take place via only partially known mechanisms. In the case of intracellular signaling, specific kinases and phosphatases interact in a coordinated manner. The dissociation of the insulin from the receptor is not sufficient to switch off the signal induced by insulin. The tyrosine kinase activity of the insulin receptor lasts as long as the regulatory domain remains phosphorylated. Shutdown takes place via cellular PTPases. Pharmacologically active compounds with an inhibitory effect on negative regulators of the insulin signal path have this
Potential, die Insulinrezeptordephosphorylierung zu verzögern. Dadurch besteht die Möglichkeit, solche Substanzen zur Verringerung der Insulinresistenz einsetzen zu können.Potential to delay insulin receptor dephosphorylation. This makes it possible to use such substances to reduce insulin resistance.
Abkürzungen:Abbreviations:
LAR Leucocyte-Antigen-Related Protein Tyrosin PhosphataseLAR Leucocyte Antigen-Related Protein Tyrosine Phosphatase
CD 45 leucocyte phosphatase CD 45CD 45 leucocyte phosphatase CD 45
YOP Yersinia protein tyrosine phosphatase PTP alpha protein tyrosine phosphatase alphaYOP Yersinia protein tyrosine phosphatase PTP alpha protein tyrosine phosphatase alpha
PTP 1B protein tyrosine phosphatase 1 BPTP 1B protein tyrosine phosphatase 1 B
TC-PTP T cell - protein tyrosine phosphataseTC-PTP T cell - protein tyrosine phosphatase
CDC-25 cell-division-control phosphatase 25CDC-25 cell division control phosphatase 25
PTEN Phosphatase (dual specific) within chromosome 10 SHP 1 ,2 src-homology phosphatase 1 ,2 Beispiele:PTEN Phosphatase (dual specific) within chromosome 10 SHP 1, 2 src-homology phosphatase 1, 2 Examples:
1. Spaltung von DiFMUP in Abhängigkeit von der PTP1 B - Enzymkonzentration:1. Cleavage of DiFMUP as a function of the PTP1 B enzyme concentration:
Die Reaktion erfolgt in einer schwarzen Mikrotiterplatte bei einer Temperatur von 37°C. Es werden pro zu analysierender Enzymkonzentration 135 μl Reaktionspuffer bereitgestellt, der die folgenden Komponenten enthält: Proteintyrosinphosphatase PTP1 B in der gewünschten Endkonzentration (Abbildung 1 : 30 - 600 ng/ ml); 50 mM Hepes pH 6,9; 150 mM NaCI; 1 mM EDTA;2 mM DTT. Die Phosphatasereaktion wird durch Zugabe von 15 μl 1 mM DiFMUP-Lösung gestartet und die Fluoreszenzzunahme (gemessen in RFU) in einem Fluoreszenz-Mikrotiterplatten-Photometer bei 358 nm Anregungs- und 455 nm Emissionswellenlänge kontinuierlich über 15 Minuten gemessen. Maß für die Enzymaktivität ist der Fluoreszenzzuwachs in Abhängigkeit von der PTP1B - Endkonzentration, welcher grafisch dargestellt werden kann (Fig. 1).The reaction takes place in a black microtiter plate at a temperature of 37 ° C. For each enzyme concentration to be analyzed, 135 μl reaction buffer is provided, which contains the following components: protein tyrosine phosphatase PTP1 B in the desired final concentration (Figure 1: 30 - 600 ng / ml); 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT. The phosphatase reaction is started by adding 15 μl of 1 mM DiFMUP solution and the increase in fluorescence (measured in RFU) is measured continuously in a fluorescence microtiter plate photometer at 358 nm excitation and 455 nm emission wavelength over 15 minutes. The measure of the enzyme activity is the increase in fluorescence as a function of the final PTP1B concentration, which can be shown graphically (FIG. 1).
2. Konzentrations-Abhängigkeit der Spaltung von DiFMUP durch PTP1 B.2. Concentration dependence of the cleavage of DiFMUP by PTP1 B.
Die Reaktion erfolgt in einer schwarzen Mikrotiterplatte bei einer Temperatur von 37°C. Es werden je 135 μl Reaktionspuffer bereitgestellt, der die folgenden Komponenten enthält: 100 ng/ ml Proteintyrosinphosphatase PTPI b; 50 mM Hepes pH 6,9; 150 mM NaCI; 1 mM EDTA;2 mM DTT. Die Phosphatasereaktion wird durch Zugabe von 15 μl DiFMUP-Lösung gestartet, die das Substrat in 10facher Konzentration der im Testansatz gewünschten Endkonzentration enthält (Abbildung 2: 0 - 200 μM), und die Fluoreszenz in einem Fluoreszenz-Mikrotiterplatten-Photometer bei 358 / 455 nm in Zeitintervallen von 30 Sekunden über 15 Minuten gemessen. Maß für dieThe reaction takes place in a black microtiter plate at a temperature of 37 ° C. 135 μl of reaction buffer are provided, which contains the following components: 100 ng / ml protein tyrosine phosphatase PTPI b; 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT. The phosphatase reaction is started by adding 15 μl DiFMUP solution, which contains the substrate in 10 times the concentration desired in the test mixture (Figure 2: 0 - 200 μM), and the fluorescence in a fluorescence microtiter plate photometer at 358/455 nm measured at intervals of 30 seconds over 15 minutes. Measure of the
Enzymaktivität ist der Fluoreszenzanstieg (gemessen in RFU) in Abhängigkeit von der DiFMUP-Konzentration, welche grafisch dargestellt werden kann (Fig. 2). Aus dieser können im Anschluß mittels Lineweaver-Burk Analyse die kinetischen Konstanten der Enzymreaktion ermittelt werden. So ergibt sich für PTP1 B ein Km-Wert von 19 μM und ein Vmax von 388000 RFU sec"1 mg"1. Analog kann diese Analyse auch für andere Tyrosinphosphatasen durchgeführt werden. Die kinetischen Konstanten können der Tabelle 1 entnommen werden. 3. Spaltung von DiFMUP in Abhängigkeit von der Enzymkonzentration der Phosphotyrosin-Phosphatasen PTPalpha, LAR, T cell-PTP, SHP-2, CD45 und YOP.Enzyme activity is the increase in fluorescence (measured in RFU) as a function of the DiFMUP concentration, which can be shown graphically (FIG. 2). From this, the kinetic constants of the enzyme reaction can then be determined using Lineweaver-Burk analysis. For PTP1 B this results in a Km value of 19 μM and a Vmax of 388000 RFU sec "1 mg " 1 . This analysis can also be carried out analogously for other tyrosine phosphatases. The kinetic constants can be found in Table 1. 3. Cleavage of DiFMUP depending on the enzyme concentration of the phosphotyrosine phosphatases PTPalpha, LAR, T cell-PTP, SHP-2, CD45 and YOP.
Die Reaktion erfolgt in einer schwarzen Mikrotiterplatte bei einer Temperatur von 37°C. Es werden pro Enzym und zu analysierender Enzymkonzentration 135 μl Reaktionspuffer bereitgestellt, der die folgenden Komponenten enthält: Proteintyrosinphosphatase in der gewünschten Endkonzentration (Fig. 3: PTPalpha: 0,5 - 1 ,85 μg/ ml, LAR: 125 - 500 ng/ ml; Tcell-PTP: 66 - 330 ng/ ml; CD 45: 50 - 400 ng/ ml; YOP: 50- 400 ng/ ml; SHP-2: 0,3 - 2,4 μg/ ml); 50 mM Hepes pH 6,9; 150 mM NaCI; 1 mM EDTA;2 mM DTT. Die Phosphatasereaktion wird durch Zugabe von 15 μl 1 mM DiFMUP-Lösung gestartet und die Fluoreszenz in einem Fluoreszenz-Mikrotiterplatten-Photometer bei 358 / 455 nm in Zeitintervallen von 30 Sekunden über 15 Minuten gemessen. Maß für die Enzymaktivität ist der Fluoreszenzanstieg (gemessen in RFU) in Abhängigkeit von der Endkonzentration der Proteintyrosin-phosphatasen , welcher grafisch dargestellt werden kann (Fig. 3).The reaction takes place in a black microtiter plate at a temperature of 37 ° C. For each enzyme and enzyme concentration to be analyzed, 135 μl reaction buffer is provided, which contains the following components: protein tyrosine phosphatase in the desired final concentration (FIG. 3: PTPalpha: 0.5-1.85 μg / ml, LAR: 125-500 ng / ml ; Tcell-PTP: 66 - 330 ng / ml; CD 45: 50 - 400 ng / ml; YOP: 50-400 ng / ml; SHP-2: 0.3 - 2.4 μg / ml); 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT. The phosphatase reaction is started by adding 15 μl of 1 mM DiFMUP solution and the fluorescence is measured in a fluorescence microtiter plate photometer at 358/455 nm in time intervals of 30 seconds over 15 minutes. The measure of the enzyme activity is the increase in fluorescence (measured in RFU) as a function of the final concentration of the protein tyrosine phosphatases, which can be shown graphically (FIG. 3).
4. Bestimmung der Hemmwirkung eines Phosphataseinhibitors von PTP1 B.4. Determination of the Inhibitory Activity of a Phosphatase Inhibitor of PTP1 B.
Der Test zur Bestimmung der Hemmwirkung des Wirkstoffes 2,2-Dioxo-2,3-dihydro- 2,6-benzo[1 ,2,3]oxathiazol-5-yl)-(9-ethyl-9H-carbazol-3- ylmethyl)-amine mittels DiFMUP-Test erfolgt in einer schwarzen Mikrotiterplatte bei einer Temperatur von 37°C. Es werden 120 μl Reaktionspuffer bereitgestellt, der die folgenden Komponenten enthält: 100 ng/ml Proteintyrosinphosphatase PTP1 B; 50 mM Hepes pH 6,9; 150 mM NaCI; 1 mM EDTA;2 mM DTT. Dazu kommen 15 μl der zu testenden Inhibitorlösung in verschiedenen Konzentrationen. DieThe test for determining the inhibitory action of the active substance 2,2-dioxo-2,3-dihydro-2,6-benzo [1, 2,3] oxathiazol-5-yl) - (9-ethyl-9H-carbazol-3- ylmethyl) -amine using the DiFMUP test is carried out in a black microtiter plate at a temperature of 37 ° C. 120 μl of reaction buffer are provided, which contains the following components: 100 ng / ml protein tyrosine phosphatase PTP1 B; 50 mM Hepes pH 6.9; 150 mM NaCl; 1mM EDTA; 2mM DTT. In addition there are 15 μl of the inhibitor solution to be tested in various concentrations. The
Phosphatasereaktion wird durch Zugabe von 15 μl 1 mM DiFMUP-Lösung gestartet, und die Fluoreszenz (gemessen in RFU) in einem Fluoreszenz-Mikrotiterplatten- Photometer bei 358 / 455 nm in Zeitintervallen von 30 Sekunden über 15 Minuten gemessen. Maß für die Enzymaktivität ist der Fluoreszenzanstieg, welcher grafisch dargestellt werden kann (Fig. 1 ). In Abhängigkeit der verwendetenPhosphatase reaction is started by adding 15 μl of 1 mM DiFMUP solution, and the fluorescence (measured in RFU) is measured in a fluorescence microtiter plate photometer at 358/455 nm at time intervals of 30 seconds over 15 minutes. The measure of the enzyme activity is the increase in fluorescence, which can be represented graphically (FIG. 1). Depending on the used
Inhibitorkonzentration ergibt sich eine Reduktion der enzymatischen Aktivität. Die Inhibitorkonzentration, bei welcher der Wirkstoffes 2,2-Dioxo-2,3-dihydro-2,6- benzo[1 ,2,3]oxathiazol-5-yl)-(9-ethyl-9H-carbazol-3- ylmethyl)-amine die Aktivität der PTP1 B um die Hälfte reduziert ( IC-50 ) kann mit 3,8 μM ermittelt werden. Zum Vergleich wurde der IC-50 Wert mit dem pNPP-Testverfahren und dem Malchitgrün- Phosphopeptidtest ermittelt. Hierbei ergibt sich für das pNPP-Testverfahren ein IC50 von 5,1 μM und für das Malchitgrün-Phosphopeptidtestverfahren ein IC50 von 3,9 μM. Die korrespondierenden Hemmkurven sind in Fig. 4 mitaufgeführt.Inhibitor concentration results in a reduction in the enzymatic activity. The inhibitor concentration at which the active ingredient 2,2-dioxo-2,3-dihydro-2,6-benzo [1, 2,3] oxathiazol-5-yl) - (9-ethyl-9H-carbazol-3-ylmethyl ) -amine the activity of PTP1 B reduced by half (IC-50) can be determined with 3.8 μM. For comparison, the IC-50 value was determined using the pNPP test method and the malchite green phosphopeptide test. This results in an IC50 of 5.1 μM for the pNPP test method and an IC50 of 3.9 μM for the malchite green phosphopeptide test method. The corresponding inhibition curves are also shown in FIG. 4.
5. Charakterisierung des Hemmtyps eines Phosphataseinhibitors auf PTPIb.5. Characterization of the inhibitor type of a phosphatase inhibitor on PTPIb.
Die Reaktion erfolgt in einer schwarzen Mikrotiterplatte bei einer Temperatur von 37°C. Es werden 120 μl Reaktionspuffer bereitgestellt, der die folgenden Komponenten enthält: 100 ng/ ml Proteintyrosinphosphatase PTPI b; 50 mM Hepes pH 6,9; 150 mM NaCI; 1 mM EDTA;2 mM DTT und einer Inhibitorkonzentration, die sich nach dem zuvor bestimmten IC50 richtet. Die Phosphatasereaktion wird durch Zugabe von 15 μl DiFMUP-Lösung gestartet, der das Substrat mit der 10fachen Konzentrationen der gewünschten Endkonzentration im Endvolumen enthält (Abbildung 2: 0 - 200 μM) und die Fluoreszenz bei in einem Fluoreszenz-Mikrotiterplatten-Photometer bei 358-455 nm in Zeitintervallen von 30 Sekunden über 15 Minuten gemessen, bis das die Reaktion in die Sättigung geht. Im Anschluß wird ein 10-facher Uberschuss der zuvor verwendeten Endkonzentration an Substrat zugesetzt und die Reaktion weiter in einem Fluoreszenz- Mikrotiterplatten-Photometer bei 358-455 nm in Zeitintervallen von 30 Sekunden über 15 Minuten verfolgt. In Gegenwart von irreversiblen Inhibitoren kann die Reaktion nicht erneut gestartet werden, bei einem reversiblen Hemmtyp ist dieses möglich (Fig. 5). 6. Charakterisierung des Hemmtyps eines Phosphataseinhibitors auf PTPI b durch zeitabhängige Inkubation.The reaction takes place in a black microtiter plate at a temperature of 37 ° C. 120 μl of reaction buffer are provided, which contains the following components: 100 ng / ml protein tyrosine phosphatase PTPI b; 50 mM Hepes pH 6.9; 150 mM NaCl; 1 mM EDTA; 2 mM DTT and an inhibitor concentration that depends on the previously determined IC50. The phosphatase reaction is started by adding 15 μl of DiFMUP solution, which contains the substrate with 10 times the desired final concentration in the final volume (Figure 2: 0 - 200 μM) and the fluorescence in a fluorescence microtiter plate photometer at 358-455 nm measured at time intervals of 30 seconds over 15 minutes until the reaction saturates. A 10-fold excess of the final concentration of substrate used previously is then added and the reaction is further monitored in a fluorescence microtiter plate photometer at 358-455 nm at time intervals of 30 seconds over 15 minutes. In the presence of irreversible inhibitors, the reaction cannot be started again; this is possible with a reversible type of inhibitor (FIG. 5). 6. Characterization of the inhibitor type of a phosphatase inhibitor on PTPI b by time-dependent incubation.
Die Reaktion erfolgt in einer schwarzen Mikrotiterplatte bei einer Temperatur von '37°C. Es werden 120 μl Reaktionspuffer bereitgestellt, der die folgendenThe reaction takes place in a black microtiter plate at a temperature of '37 ° C. 120 ul reaction buffer is provided, the following
Komponenten enthält: 100 ng/ ml Proteintyrosinphosphatase PTPIb; 50 mM Hepes pH 6,9; 150 mM NaCI; 1 mM EDTA;2 mM DTT und einer Inhibitorkonzentration, die sich nach dem zuvor bestimmten IC50 richtet.Component contains: 100 ng / ml protein tyrosine phosphatase PTPIb; 50 mM Hepes pH 6.9; 150 mM NaCl; 1 mM EDTA; 2 mM DTT and an inhibitor concentration that depends on the previously determined IC50.
Der Ansatz wird inkubiert und zu definierten Zeitpunkten die Phosphatasereaktion durch Zugabe von 15μl DiFMUP-Lösung gestartet, der das Substrat mit der 10fachen Konzentrationen der gewünschten Endkonzentration im Endvolumen enthält (Abbildung 2: 0 - 200 μM) und die Fluoreszenz bei in einem Fluoreszenz- Mikrotiterplatten-Photometer bei 358-455 nm in Zeitintervallen von 30 Sekunden über 15 Minuten gemessen. In Gegenwart von irreversiblen Inhibitoren kommt es in Abhängigkeit von der Vorinkubationszeit zu einer Verringerung der Enzymaktivität, während dieses bei Inhibitoren mit reversiblem Hemmtyp nicht beobachtet werden kann (Fig. 6).The mixture is incubated and the phosphatase reaction started at defined times by adding 15μl DiFMUP solution, which contains the substrate with 10 times the concentration of the desired final concentration in the final volume (Figure 2: 0 - 200 μM) and the fluorescence in a fluorescence microtiter plate -Photometer measured at 358-455 nm in time intervals of 30 seconds over 15 minutes. In the presence of irreversible inhibitors there is a decrease in enzyme activity depending on the preincubation time, whereas this cannot be observed in the case of inhibitors with a reversible inhibitor type (FIG. 6).
Verzeichnis der Abbildungen:List of pictures:
Fig. 1 : Spaltung von DiFMUP in Abhängigkeit von der PTP1 B - Enzymkonzentration.Fig. 1: Cleavage of DiFMUP depending on the PTP1 B enzyme concentration.
Fig. 2: Konzentrations-Abhängigkeit der Spaltung von DiFMUP durch PTP1B.Fig. 2: concentration dependence of the cleavage of DiFMUP by PTP1B.
Fig. 3: Spaltung von DiFMUP in Abhängigkeit von der Enzymkonzentration der Phosphotyrosin-Phosphatasen PTPalpha, LAR, TCPTP, SHP 2, CD45 und YOP.3: Cleavage of DiFMUP as a function of the enzyme concentration of the phosphotyrosine phosphatases PTPalpha, LAR, TCPTP, SHP 2, CD45 and YOP.
Fig. 4: Bestimmung der Hemmwirkung eines Phosphataseinhibitors von PTP1B.Fig. 4: Determination of the inhibitory effect of a phosphatase inhibitor of PTP1B.
Fig. 5: Charakterisierung des Hemmtyps eines Phosphataseinhibitors5: Characterization of the inhibition type of a phosphatase inhibitor
Abb.6: Charakterisierung des Hemmtyps eines Phosphataseinhibitors durch zeitabhängige Inkubation Fig.6: Characterization of the inhibitor type of a phosphatase inhibitor by time-dependent incubation

Claims

Patentansprüche claims
1. Verfahren zum Nachweis der enzymatischen Aktivität einer Protein-Tyrosin- Phosphatase in biologischem Material, wobei1. A method for the detection of the enzymatic activity of a protein tyrosine phosphatase in biological material, wherein
a] biologisches Material oder eine Präparation aus biologischem Material bereitgestellt wird,a] biological material or a preparation made of biological material is provided,
b] 6,8-Difluoro-4-methylumbelliferylphosphat (DiFMUP) bereitgestellt wird,b] 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) is provided,
c] das biologische Material oder die Präparation aus biologischem Material aus a] mit dem DiFMUP aus b] in einer wäßrigen Lösung in Kontakt gebracht wird,c] the biological material or the preparation of biological material from a] is brought into contact with the DiFMUP from b] in an aqueous solution,
d] das dann entstehende 6,8-Difluoro-4-methylumbelliferyl fluorometrisch nachgewiesen wird.d] the resulting 6,8-difluoro-4-methylumbelliferyl is detected fluorometrically.
2. Verfahren nach Anspruch 1 , wobei als Präparation aus biologischem Material wenigstens eine Protein Tyrosin Phosphatase aus der Gruppe LAR, CD 45, YOP, PTP alpha, PTP 1 B, TC-PTP, CDC 25, PTEN, SHP1 ,2 bereitgestellt wird.2. The method according to claim 1, wherein at least one protein tyrosine phosphatase from the group LAR, CD 45, YOP, PTP alpha, PTP 1 B, TC-PTP, CDC 25, PTEN, SHP1, 2 is provided as a preparation from biological material.
3. Verfahren nach Anspruch 1 oder 2, wobei die Konzentration des DiFMUP nach Inkontaktbringen 10 - 250 μM beträgt.3. The method according to claim 1 or 2, wherein the concentration of the DiFMUP after contacting is 10-250 μM.
4. Verfahren nach Anspruch 3, wobei die Konzentration des DiFMUP 50 bis 100 μM beträgt.4. The method according to claim 3, wherein the concentration of the DiFMUP is 50 to 100 μM.
5. Verfahren nach einem oder mehreren der Ansprüche 1 bis 4, wobei der pH-Wert der wäßrigen Lösung in c] zwischen 5,0 und 8,0 liegt.5. The method according to one or more of claims 1 to 4, wherein the pH of the aqueous solution in c] is between 5.0 and 8.0.
6. Verfahren nach Anspruch 5, wobei der pH-Wert zwischen 6,0 und 7,5 liegt. 6. The method of claim 5, wherein the pH is between 6.0 and 7.5.
7. Verfahren nach Anspruch 5 und 8, wobei der pH-Wert 7,0 beträgt.7. The method according to claim 5 and 8, wherein the pH is 7.0.
8. Verfahren zur Identifizierung einer Verbindung, welche die Aktivität einer Protein-Tyrosin-Phosphatase modifiziert, wobei8. A method of identifying a compound that modifies the activity of a protein tyrosine phosphatase, wherein
a] eine chemische Verbindung bereitgestellt wird, b] biologisches Material oder eine Präparation aus biologischem Material bereitgestellt wird, c] 6,8-Difluoro-4-methylumbelliferylphosphat (DiFMUP) bereitgestellt wird, d] die chemische Verbindung aus a], sowie das biologische Material oder diea] a chemical compound is provided, b] biological material or a preparation of biological material is provided, c] 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) is provided, d] the chemical compound from a], as well as the biological Material or the
Präparation aus biologischem Material aus b] und das DiFMUP aus c] miteinander in einer wäßrigen Lösung in Kontakt gebracht werden, e] die Menge des dann entstehenden 6,8-Difluoro-4-methylumbelliferyl fluorometrisch bestimmt wird, f] die Menge des entstehenden 6,8-Difluoro-4-methylumbelliferyl aus e] verglichen wird mit der Menge des entstehenden 6,8-Difluoro-4 methylumbelliferyl in einem Kontrollansatz.Preparation of biological material from b] and the DiFMUP from c] are brought into contact with one another in an aqueous solution, e] the amount of the resulting 6,8-difluoro-4-methylumbelliferyl is determined fluorometrically, f] the amount of the resulting 6 , 8-difluoro-4-methylumbelliferyl from e] is compared with the amount of the resulting 6,8-difluoro-4 methylumbelliferyl in a control batch.
9. Verfahren nach Anspruch 8, wobei die Aktivität einer Protein-Tyrosin- Phosphatase stimuliert, gehemmt oder aufrechterhalten wird.9. The method of claim 8, wherein the activity of a protein tyrosine phosphatase is stimulated, inhibited or maintained.
10. Verfahren nach Anspruch 8 oder 9, wobei die Protein-Tyrosin-Phosphatase aus der Gruppe LAR, CD 45, YOP, PTP alpha, PTP 1 B, TC-PTP, CDC 25, PTEN, SHP1.2 ausgewählt wird.10. The method according to claim 8 or 9, wherein the protein tyrosine phosphatase is selected from the group LAR, CD 45, YOP, PTP alpha, PTP 1 B, TC-PTP, CDC 25, PTEN, SHP1.2.
11. Verbindung identifiziert durch ein Verfahren gemäß Anspruch 8 bis 10.11. Connection identified by a method according to claims 8 to 10.
12.Verbindung nach Anspruch 11 , wobei die Masse der Verbindung zwischen 0,1 und 50 kDa liegt.12.A compound according to claim 11, wherein the mass of the connection is between 0.1 and 50 kDa.
13. Verbindung nach Anspruch 11 oder 12, wobei die Masse der Verbindung zwischen 0,1 und 5 kDa liegt. 13. A compound according to claim 11 or 12, wherein the mass of the compound is between 0.1 and 5 kDa.
14. Verbindung nach Anspruch 11 bis 13, wobei die Masse der Verbindung zwischen 0,1 und 3 kDa liegt.14. The compound of claim 11 to 13, wherein the mass of the connection is between 0.1 and 3 kDa.
5 15. Verbindung nach einem oder mehreren der Ansprüche 11 bis 14, wobei die Verbindung ein Protein, eine Aminosäure, ein Polysaccharid, ein Zucker, ein Polynukleotid, ein Nukleotid, ein Naturstoff oder eine aromatische Kohlenwasserstoffverbindung ist.15. A compound according to one or more of claims 11 to 14, wherein the compound is a protein, an amino acid, a polysaccharide, a sugar, a polynucleotide, a nucleotide, a natural product or an aromatic hydrocarbon compound.
10 16. Arzneimittel enthaltend mindestens eine Verbindung gemäß Anspruch 11 bis 15, Formulierungshilfsstoffe für ein Arzneimittel und/oder polymere Zusatzstoffe.16. Medicaments containing at least one compound according to claims 11 to 15, formulation auxiliaries for a medicament and / or polymeric additives.
17. Verwendung einer Verbindung nach einem oder mehreren der Ansprüche 11 bis 14 zur Herstellung eines Arzneimittels zur Behandlung von Diabetes. 15 17. Use of a compound according to one or more of claims 11 to 14 for the manufacture of a medicament for the treatment of diabetes. 15
EP02796734A 2002-01-04 2002-12-24 Highly sensitive and continuous protein-tyrosine-phosphatase (ptpase) test using 6.8 difluoro-4-methyl-umbelliferylphosphate Withdrawn EP1466009A2 (en)

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DE10200173 2002-01-04
DE2002100173 DE10200173A1 (en) 2002-01-04 2002-01-04 Detecting activity of protein-tyrosine phosphatase by detecting hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate, useful for identifying modulators of the enzyme that can be used to treat diabetes
DE2002136329 DE10236329A1 (en) 2002-08-08 2002-08-08 Detecting activity of protein-tyrosine phosphatase by detecting hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate, useful for identifying modulators of the enzyme that can be used to treat diabetes
DE10236329 2002-08-08
PCT/EP2002/014755 WO2003056029A2 (en) 2002-01-04 2002-12-24 Highly sensitive and continuous protein-tyrosine-phosphatase (ptpase) test using 6,8 difluoro-4-methyl-umbelliferylphosphate

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US8008035B2 (en) 2007-01-25 2011-08-30 Roche Diagnostics Operations, Inc. Enhancement of vanadium-containing phosphatase inhibitors
EP1949894B1 (en) * 2007-01-25 2016-06-29 Roche Diagnostics GmbH Enhancement of vanadium-containing phosphatase inhibitors by polyols
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