WO2003056029A2 - Test de detection a haute sensibilite et en continu d'une proteine tyrosine phosphatase (ptpase) au moyen de 6,8-difluoro-4-methyl-ombelliferylphosphate - Google Patents

Test de detection a haute sensibilite et en continu d'une proteine tyrosine phosphatase (ptpase) au moyen de 6,8-difluoro-4-methyl-ombelliferylphosphate Download PDF

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Publication number
WO2003056029A2
WO2003056029A2 PCT/EP2002/014755 EP0214755W WO03056029A2 WO 2003056029 A2 WO2003056029 A2 WO 2003056029A2 EP 0214755 W EP0214755 W EP 0214755W WO 03056029 A2 WO03056029 A2 WO 03056029A2
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WIPO (PCT)
Prior art keywords
biological material
compound
difluoro
difmup
protein tyrosine
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PCT/EP2002/014755
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German (de)
English (en)
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WO2003056029A3 (fr
Inventor
Stefan Welte
Norbert Tennagels
Stefan Petry
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Aventis Pharma Deutschland Gmbh
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Priority claimed from DE2002100173 external-priority patent/DE10200173A1/de
Priority claimed from DE2002136329 external-priority patent/DE10236329A1/de
Priority to KR10-2004-7010451A priority Critical patent/KR20040073539A/ko
Priority to CA002471601A priority patent/CA2471601A1/fr
Priority to IL16283202A priority patent/IL162832A0/xx
Priority to AU2002361217A priority patent/AU2002361217A1/en
Application filed by Aventis Pharma Deutschland Gmbh filed Critical Aventis Pharma Deutschland Gmbh
Priority to MXPA04006361A priority patent/MXPA04006361A/es
Priority to JP2003556546A priority patent/JP2005512600A/ja
Priority to EP02796734A priority patent/EP1466009A2/fr
Publication of WO2003056029A2 publication Critical patent/WO2003056029A2/fr
Publication of WO2003056029A3 publication Critical patent/WO2003056029A3/fr
Priority to NO20043244A priority patent/NO20043244L/no

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—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

Abstract

L'invention concerne un procédé permettant de déceler une protéine tyrosine phosphatase dans une matière biologique au moyen de 6,8-difluoro-4-méthyl-ombelliférylphosphate (DiFMUP).
PCT/EP2002/014755 2002-01-04 2002-12-24 Test de detection a haute sensibilite et en continu d'une proteine tyrosine phosphatase (ptpase) au moyen de 6,8-difluoro-4-methyl-ombelliferylphosphate WO2003056029A2 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
EP02796734A EP1466009A2 (fr) 2002-01-04 2002-12-24 Test de detection a haute sensibilite et en continu d'une proteine tyrosine phosphatase (ptpase) au moyen de 6,8-difluoro-4-methyl-ombelliferylphosphate
JP2003556546A JP2005512600A (ja) 2002-01-04 2002-12-24 ６，８−ジフルオロ−４−メチルウンベリフェリルホスフェートを用いた高感度連続的蛋白質チロシンホスファターゼ（ＰＴＰａｓｅ）試験
CA002471601A CA2471601A1 (fr) 2002-01-04 2002-12-24 Test de detection a haute sensibilite et en continu d'une proteine tyrosine phosphatase (ptpase) au moyen de 6,8-difluoro-4-methyl-ombelliferylphosphate
IL16283202A IL162832A0 (en) 2002-01-04 2002-12-24 Highly sensitive and continous protein-tyrosine-phosphatase (ptpase) test using 6,8 difluoro-4-methylumbe
AU2002361217A AU2002361217A1 (en) 2002-01-04 2002-12-24 Highly sensitive and continuous protein-tyrosine-phosphatase (PTPase) test using 6,8 difluoro-4-methyl-umbelliferylphosphate
KR10-2004-7010451A KR20040073539A (ko) 2002-01-04 2002-12-24 6,8-디플루오로-4-메틸-움벨리페릴포스페이트를 사용하는고감수성의 연속적단백질-티로신-포스파타제(ＰＴＰａｓｅ) 시험
MXPA04006361A MXPA04006361A (es) 2002-01-04 2002-12-24 Un ensayo de deteccion de proteinas del tipo tirosina fosfatasas (ptpasas) continuo y altamente sensible que usa fosfato de 6,8-difluoro-4-metilumbeliferilo.
NO20043244A NO20043244L (no) 2002-01-04 2004-08-02 Meget folsom og kontinuerlig protein-tyrosin-fosfatase (PTPASE) test ved anvendelse av 6,8-difluor-4-metyl-umbelliferylfosfat (DiFMUP)

Applications Claiming Priority (4)

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DE10200173.1 2002-01-04
DE2002100173 DE10200173A1 (de) 2002-01-04 2002-01-04 Ein hochsensitiver und kontinuerlicher Protein Tyrosin-Phosphatase (PTPase) Test unter Verwendung von 6,8 Difluoro-4-methyl-umbelliferylphosphat (DiFMUP)
DE10236329.3 2002-08-08
DE2002136329 DE10236329A1 (de) 2002-08-08 2002-08-08 Ein hochsensitiver und kontinuierlicher Protein Tyrosin-Phosphatase (PTPase) Test unter Verwendung von 6,8 Difluoro-4-methyl-umbelliferylphosphat (DiFMUP)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100350045C (zh) * 2003-08-21 2007-11-21 北京农业生物技术研究中心 玉米酪氨酸蛋白磷酸酶基因及其编码蛋白与应用
EP1949894A3 (fr) * 2007-01-25 2011-12-28 Roche Diagnostics GmbH Amélioration des inhibiteurs de phosphatase contenant du vanadium

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8008035B2 (en) 2007-01-25 2011-08-30 Roche Diagnostics Operations, Inc. Enhancement of vanadium-containing phosphatase inhibitors
CN113155787A (zh) * 2020-12-29 2021-07-23 武汉合研生物医药科技有限公司 一种突变酶shp2 e76k的活性检测方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061410A1 (fr) * 1998-05-12 1999-12-02 American Home Products Corporation Diphenyles 2,3,5-substitues utiles pour le traitement de la resistance insulinique et de l'hyperglycemie

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061410A1 (fr) * 1998-05-12 1999-12-02 American Home Products Corporation Diphenyles 2,3,5-substitues utiles pour le traitement de la resistance insulinique et de l'hyperglycemie

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GOLDSTEIN B J: "Protein-tyrosine phosphatase 1B (PTP1B): a novel therapeutic target for type 2 diabetes mellitus, obesity and related states of insulin resistance." CURRENT DRUG TARGETS. IMMUNE, ENDOCRINE AND METABOLIC DISORDERS. NETHERLANDS NOV 2001, Bd. 1, Nr. 3, November 2001 (2001-11), Seiten 265-275, XP001153562 ISSN: 1568-0088 *
KERBY M ET AL: "A fluorogenic assay using pressure-driven flow on a microchip." ELECTROPHORESIS. GERMANY OCT 2001, Bd. 22, Nr. 18, Oktober 2001 (2001-10), Seiten 3916-3923, XP001153559 ISSN: 0173-0835 *
MALAMAS M S ET AL: "Novel Benzofuran and Benzothiophene Biphenyls as Inhibitors of Protein Tyrosine Phophatase 1B with Antihyperglycemic Properties" JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, Bd. 43, Nr. 7, 6. April 2000 (2000-04-06), Seiten 1293-1310, XP002190818 ISSN: 0022-2623 *
MATTER WILLIAM F ET AL: "Role of PRL-3, a human muscle-specific tyrosine phosphatase, in angiotensin-II signaling." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Bd. 283, Nr. 5, 25. Mai 2001 (2001-05-25), Seiten 1061-1068, XP002247489 ISSN: 0006-291X *
WROBEL JAY ET AL: "PTP1B inhibition and antihyperglycemic activity in the ob/ob mouse model of novel 11-arylbenzo(b)naphtho(2,3-d)furans and 11-arylbenzo(b)naphtho(2,3-d)thiophenes" JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, Bd. 42, Nr. 17, 26. August 1999 (1999-08-26), Seiten 3199-3202, XP002181876 ISSN: 0022-2623 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100350045C (zh) * 2003-08-21 2007-11-21 北京农业生物技术研究中心 玉米酪氨酸蛋白磷酸酶基因及其编码蛋白与应用
EP1949894A3 (fr) * 2007-01-25 2011-12-28 Roche Diagnostics GmbH Amélioration des inhibiteurs de phosphatase contenant du vanadium

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NO20043244L (no) 2004-08-02
CA2471601A1 (fr) 2003-07-10
RU2004123794A (ru) 2005-04-20
MXPA04006361A (es) 2004-10-04
EP1466009A2 (fr) 2004-10-13
CN1612939A (zh) 2005-05-04
WO2003056029A3 (fr) 2004-04-01
AU2002361217A1 (en) 2003-07-15
BR0215452A (pt) 2004-11-23
KR20040073539A (ko) 2004-08-19
JP2005512600A (ja) 2005-05-12

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