KR20040073539A - Highly sensitive and continuous protein-tyrosine-phosphatase(PTPase) test using 6,8-difluoro-4-methyl-umbelliferylphosphate - Google Patents

Abstract

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

Description

Highly sensitive and continuous protein-tyrosine-phosphatase test using 6,8-difluoro-4-methyl-umbelliferylphosphate -difluoro-4-methyl-umbelliferylphosphate}

The present invention relates to an improved method of detecting protein-tyrosine-phosphatase, in particular to an improved method of detecting protein-tyrosine-phosphatase using 6,8-difluoro-4-methylumbelliferylphosphate under neutral conditions. will be.

The prior art describes methods for detecting the activity of protein-tyrosine-phosphatase using the substrate p-nitrophenyl phosphate (p-NPP). Such detection methods are described in Standard Biochemistry Handbook ["Current protocols in protein science: John E. Coligan (ed.), Ben M. Dunn (ed.), Hidde L. Ploegh (ed.), David W. Speicher (ed. .), Paul T. Wingfield (ed.); SBN: 0-471-11184-8; loose-leaf pages, continuously updated; published by John Wiley & Sons. ". The action of phosphatase produces p-nitrophenol from p-NPP. p-nitrophenol can be detected by photometry based on a dark yellow color in the alkaline range.

However, this test method has some disadvantageous features. This test is not suitable for directly determining the activity of an enzyme because it is performed according to the time-stop principle. According to this principle, the enzymatic reaction is stopped by the addition of sodium hydroxide solution after a predetermined time. As the pH increases, the color of the resulting p-nitrophenol turns yellow, the absorption of which is determined by photometry and is a measure of the amount of p-nitrophenol present. This test principle is somewhat scrutinized for the investigation of enzyme-motion, for example to determine the type of inhibition. Although a number of experimental guidelines are required, a corresponding number of individual analyzes that are coordinated with each other must be established.

In addition, the substrate is sensitive to light, temperature and pH. Within the physiological pH range, the substrate tends to degrade more slowly.

When pNPP is used as the substrate, some of the tyrosine phosphatase investigated has the maximum activity present in the acid range. For example, PTP 1B has a high conversion at pH 5.6. On the other hand, at physiological pH 7.0, PTP 1B only works at 30% of the maximum conversion value when using pNPP as the substrate. This necessitates the use of relatively high quality enzymes, which leads to a corresponding increase in cost. Another negative factor of this assay is that other components present in the test, such as buffers, salts or other substances (test substances), are sometimes absorbed in the yellow range. This requires proper background control.

The malachite green phosphopeptide test has been known as another method for detecting the activity of protein-tyrosine-phosphatase. See Martin et al., (1985) Journal of Biological Chemistry, 260, pp. 14932 and Harder et al., (1994) Biochemical Journal, 298, pp. 395]. In this method, inorganic phosphate that releases phosphatase from its peptide substrate is detected by photometry using malachite green reagent. Apart from the temporal nature of the photometric crystals resulting from impurities or pH sensitivity and the difficulty of time-stopping, for example, another drawback is that certain peptide substrates must be prepared in high concentrations for each phosphatase, The fact generally makes the method somewhat expensive.

3,6-Fluorescein diphosphate has been described as another substrate for detecting protein-tyrosine-phosphatase (Journal of Biomolecular Screening 4, 327-334, 1999). Such substrates, in contrast to the two substrates mentioned above, allow for direct measurement of enzymatic activity, but the spectral properties of these substrates are still not particularly advantageous for performing measurements in the physiological pH range.

Accordingly, the object of the present invention relates to an improved method for detecting another commercially available protein-tyrosine-phosphatase.

The present invention

Providing a biological material or an agent obtained from the biological material (a),

(B) providing 6,8-difluoro-4-methylumbelliferylphosphate (DiFMUP),

(C) contacting the biological material from step (a) or a preparation obtained from the biological material with DiFMUP from step (b) in an aqueous solution, and

A method for detecting the enzymatic activity of protein-tyrosine-phosphatase in a biological material, comprising the step (d) of detecting subsequently generated 6,8-difluoro-4-methylumbelliferyl by fluorometry. It is about.

Formulations obtained from biological materials preferably comprise protein-tyrosine-phosphatase selected from the groups LAR, CD 45, PTP α, PTP 1B, TC-PTP, YOP, CDC 25, PTEN and SHP1,2. Agents obtained from biological materials may be present in steps of differing purity. The agent obtained from the biological material may be a sample rich in whole cells, lysed cells, cellular components and / or organelles, or purified proteins.

6,8-difluoro-4-methylumbelliferylphosphate (DiFMUP) when contacted with a biological material or an agent obtained from a biological material, 6,8-difluoro-4-methylumbeliferyl phosphate (DiFMUP) ) Concentration is preferably 10 to 250 μM. Especially preferably, the concentration of DiFMUP is 50-100 μM.

The pH of the aqueous solution in which the biological material or the preparation obtained from the biological material is contacted with DiFMUP is preferably 5.0 to 8.0, particularly preferably 6.0 to 7.5. In even more particularly preferred embodiments, the pH is 7.0.

The invention also

Providing a chemical compound (a),

(B) providing a biological material or an agent obtained from the biological material,

(C) providing 6,8-difluoro-4-methylumbelliferylphosphate (DiFMUP),

(D) contacting each other in an aqueous solution with the chemical compound from step (a), the biological material from step (b) or the agent obtained from the biological material and DiFMUP from step (c),

(E) quantitatively determining the amount of 6,8-difluoro-4-methylumbelliferyl produced subsequently by fluorometry and

Comparing the amount of 6,8-difluoro-4-methylumbelliferyl produced from step (e) with the amount of 6,8-difluoro-4-methylumbelliferyl produced by a control assay (f) It relates to a method for identifying a compound that changes the activity of protein-tyrosine-phosphatase, including.

In particular, control assays, when contacting a biological material or an agent obtained from a biological material with DiFMUP, do not include a chemical compound that is not included in the concept of treatment step (a) previously mentioned or is associated with a selected type of protein-tyrosine-phosphatase. It is characterized by the fact that the effects of the compounds are already known. Such chemical compounds which are used in control assays and affect protein-tyrosine-phosphatase are already known, in particular vanadate, vanadium organic compounds, vanadate, okadaic acid, NaF, depostascin, modified peptides or other Compound.

In various preferred embodiments of the methods of identifying compounds that change the activity of protein-tyrosine-phosphatase, such improvements should include the stimulation, inhibition or stability of the activity of protein-tyrosine-phosphatase.

In another preferred embodiment of the method of identifying compounds that alter the activity of protein-tyrosine-phosphatase, such protein-tyrosine-phosphatase is selected from the group LAR, CD 45, PTP α, TC-PTP, CDC 25, PTEN, YOP, It is selected from the group consisting of SHP1, 2 and PTP 1B.

The present invention also relates to compounds identified by the above-mentioned methods for identifying compounds that change the activity of protein-tyrosine-phosphatase and change the activity of protein-tyrosine-phosphatase. The mass of this compound is preferably 0.1 to 50 kDa, more preferably 0.1 to 5 kDa, even more preferably 0.1 to 3 kDa. This compound may be a protein, amino acid, polynucleotide, nucleotide, natural product or aromatic hydrocarbon compound. The present invention also relates to a medicament comprising at least one compound as mentioned above, which compound is identified as a method for identifying compounds that alter the activity of protein-tyrosine-phosphatase. The medicament further comprises an auxiliary substance for formulating the medicament and / or the polymerizable additive.

The invention also relates to the use of a compound identified as a method for identifying a compound that alters the activity of protein-tyrosine-phosphatase for the manufacture of a medicament for the treatment of diabetes.

6,8-difluoro-4-methylumbelliferyl phosphatase is commercially available. For example, Molecular Probes Europe BV (Leiden, Netherlands 2333) sells this product. Its preparation is described in US Pat. No. 5,830,912.

A biological substance is any substance that contains genetic information and itself is also a bacterium or fungus such as Escherichia coli or Saccharomyces cerevisiae. Biological materials also include cells from cell media.

In the case of cells from animal or human tissues, biological materials can be obtained by biopsy, surgical excision, excision with a syringe or catheter or comparable techniques. Cells excised in this manner can be severely frozen, post-treated or introduced into the medium. Bacteria and yeast cells can be propagated using conventional microbiological techniques and workup.

Those skilled in the art for this purpose are well known in the literature "Current Protocols in Molecular Biology; ed .: FM Ansabel et al., Loose-leaf publication, continuously updated, 2001 edition, published by John Wiley & Sons". Find instructions. The biological material may also include cells from the medium of the animal cells. Examples of such cells are mouse cells, rat cells or hamster cells. Cell Medium Cells can be of primary cell type or established cell line. Examples of established cell lines are mouse 3T3 cells, CHO cells or Hela cells. The maintenance, growth and propagation of cell lines is described in standard textbooks ("Basic Cell Culture; ed .: J.M. Daris IRL Press, Oxford (1996))."

Preparations obtained from biological materials are prepared, for example, by the destruction of the biological material and subsequent purification steps. The method of destroying the biological material may in particular be repeated freezing, thawing, sonication, the use of a French press or the addition of detergents and enzymes. Subsequent purification steps include, for example, differential centrifugation, precipitation with ammonium sulfate or an organic solvent, use of chromatographic techniques, and the like. Examples of chromatographic techniques include polyacrylamide gel electrophoresis, high pressure liquid chromatography, ion exchange chromatography, affinity chromatography, gas chromatography, and mass spectrometry. Textbooks, such, in particular, "Current Protocols in Protein Science, ed .: JE Coligan et al., Loose-leaf publication, continuously updated, 2001 edition, published by John Wiley & Sons" serve this purpose. It can be used by the skilled person for the sake of detail and in particular for the purification of the protein.

Biological materials or preparations obtained from biological materials can be contacted with DiFMUP in conventional laboratory containers such as Eppendorf tubes, centrifuge tubes or glass flasks. Inherent aqueous media include, for example, buffer substances, nutrient media components, single charged or double charged ions (eg, Na ⁺ , K ⁺ , Ca ²⁺ , Cl ⁻ , SO ₄ ^2- and PO ₃ ^2-). ) Or other ions and further proteins, glycerol or other substances. For contacting, particular conditions may be advantageous, in particular, such as temperature, pH, ionic conditions, protein concentration, volume or other factors. This is achieved, for example, by using ions or proteins in the amounts previously weighed correctly or by contacting them in a culture apparatus maintained at a constant temperature in the presence of a buffer. The aqueous solvent may in particular contain a certain proportion of organic solvents such as dimethyl sulfoxide, methanol or ethanol. However, the content of such solvents is preferably 10% by volume or less of the mixture.

The PTPase (phosphatase) protein group comprises about 100 different members simultaneously. This absence is roughly subdivided into receptor-bound proteins and cytoplasmic proteins. Phosphatase commonly has the amino acid motif (H / V) CX ₅ R (S / T) in the catalytic domain. Receptor bound phosphatase typically consists of an extracellular domain, a single membrane region and one or two cytoplasmic PTPase domains. LAR (Leukocyte Common Antigen Related) protein and PTPα protein are thought to belong to receptor bound PTPase. Intracellular PTPases generally comprise various extensions and catalytic domains of the C-terminal or N-terminal region, for example as a result of the "SH domain. Such extensions are believed to have the desired or regulating function. Enzyme PTP 1B is defined as cytoplasmic PTPase. In addition to pure tyrosine phosphatase, the PTPase group also includes a double phosphatase group. In addition to phosphotyrosine, such enzymes also use phosphoserine or phospholeonin as substrates. Such groups include, for example, phosphatase VHR and cdc25.

Phosphatase LAR, PTPα, SHP-2 and PTP 1B are believed to have important functions in the insulin mediated signaling pathway. These PTPases bind to insulin receptors and catalyze dephosphorylation. Such PTPases may act individually or in combination, possibly in the pathogenesis of insulin resistance. Biochemistry 38, 3793-3803, 1999; p. 3799].

PTP 1B is a negative regulator of the insulin stimulus signaling pathway, ie the protein once again converts the signal induced by insulin. Perhaps the signaling pathway is blocked by insulin receptors that are directly dephosphorylated. PTP 1B is also aberrantly expressed in most patients with breast cancer. In addition, enzymes interact with "epidermal growth factor". The enzyme was demonstrated to have two aryl phosphate binding pockets. One is located directly at the active center and the other is located outside of it adjacent to the catalyst center (Biochemistry 38, 3793-3803, 1999).

The transmission and termination of many intracellular signals is regulated by tyrosine-phosphorylation of the factors involved. Exactly balanced activity of complementary protein tyrosine kinases (PTK) and phosphatase (PTPase), particularly protein-tyrosine-phosphatase, establishes phosphorylation status.

As an enzyme, PTPase results in selective dephosphorylation of phosphotyrosine residues. Interactions with protein tyrosine kinases, various different biological processes, and PTPase function in the mediation of signals are due to, for example, growth factors or hormones. Such signal transduction mechanisms play an important role in regulating cellular metabolism, growth, differentiation or mobility.

Incomplete regulation of the signal pathway is thought to be one cause of many pathological processes. Such processes include, for example, cancer, some immunological and neurological diseases, and type II diabetes and obesity.

Chemical compounds are provided, for example, by chemical synthesis. Standard synthesis methods are well known to the skilled person. The chemical compound may be part of the collected chemical compound, as produced by storing and cataloging the chemical compound from a terminating synthetic program (called a chemical library). In other cases, chemical compounds may be produced by microorganisms, in particular bacteria or other fungi or plants (natural products).

Pharmaceutical compounds suitable for oral administration may be present in discrete units such as capsules, cachets, suction tablets or tablets as powders or granules, as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil emulsions. have. Such compositions may be prepared according to any suitable pharmaceutical method comprising contacting the active compound (which may be composed of one or more additional ingredients) and excipients. Generally, such compositions are prepared by, if necessary, after the product is formed, the active compound is uniformly and homogeneously mixed with the liquid and / or finely divided solid excipients.

Thus, tablets may be prepared, optionally with one or more additional ingredients, for example by pressing or forming a powder or granules of the compound.

Compressed tablets can be prepared by tableting the compounds into free flowing forms such as powders or granules when mixed with binders, glidants, inert diluents and / or (several) surfactants / dispersants in a suitable machine. The tablets formed can be prepared by forming a powder compound that is moisturized with an inert liquid diluent in a suitable machine.

Pharmaceutical compositions suitable for oral (sublingual) administration include lozenges comprising the compound in a perfume substance, conventional sucrose and gum arabic or tragacanth, and inert bases such as gelatin and glycerol or sucrose and arivia gum. Together with suction tablets containing the compound.

Pharmaceutical compositions suitable for parenteral administration preferably comprise sterile aqueous formulations of the compound, which formulation is preferably isotonic by the blood of the intended recipient. Such formulations are preferably administered intravenously, even when subcutaneous, intramuscular or intradermal administration is possible by injection. Such formulations may preferably be prepared by mixing the compound with water and making the resulting solution sterile and isotonic by the blood. Injectable compositions according to the invention generally comprise 0.1 to 5% by weight of active compound.

Pharmaceutical compositions suitable for rectal administration are preferably in the form of single dose suppositories.

Such pharmaceutical compositions can be prepared by mixing the compound with one or more conventional solid excipients such as cocoa butter and forming the resulting mixture.

Pharmaceutical compositions suitable for topical use on the skin are preferably present in the form of ointments, creams, lotions, pastes, sprays, aerosols or emulsions. Excipients that can be used are petrolatum, lanolin, polyethylene glycol, alcohols and mixtures of two or more of these substances. The active compound is generally present at a concentration of 0.1 to 15% by weight, for example 0.5 to 2% by weight of the composition.

Transdermal administration is also possible. Pharmaceutical compositions suitable for transdermal use may be present as discrete gypsum suitable for hermetic contact and long term contact with the epidermis of the patient. Such gypsum suitably comprises the active compound in an aqueous solution which is buffered, optionally dissolved and / or dispersed in an adhesive or dispersed in a polymer. Suitable concentrations of the active compound are about 1 to 35%, preferably about 3 to 15%.

Type II diabetes mellitus (NIDDM-non-insulin dependent diabetes mellitus) is characterized by high glucose values (polysaccharide) in fasting (less than 126 mg / dl), insulin resistance in peripheral tissues such as muscle or fat, increased uveogenesis in the liver, and Improper secretion of insulin by pancreatic β cells. The actual cause of this disease is not yet known. Type II diabetes occurs very often with other clinical manifestations such as obesity, hypertriglyceridemia (elevated blood fat levels) and hypertension.

Insulin resistance is believed to be the key to understanding the clinical picture. Insulin resistance is expressed upon decreased ability of peripheral organs and responds to a predetermined insulin concentration. This is reflected at the cellular level, i.e., when the amount of insulin required to induce an effect due to insulin is increased. In muscle, fat and liver cells, insulin has many effects on glucose metabolism and fat metabolism, such as increasing glucose uptake from the blood, increasing the rate of glucose metabolism or inhibiting fatty acid degradation. Various factors are thought to be fundamentally related to the development of insulin resistance at the cellular level. Insulin receptors, signal cascade factors and components of the glucose delivery system play an important role in this regard.

Insulin exerts its biological function by binding to the insulin receptor in the first step. After this insulin receptor binds to insulin, its β subunit is autophosphorylated by insulin receptor kinase. In muscle cells, the signal is delivered by IRS (insulin receptor substrate) and PI3K (phosphoinositol-3-kinase) and induces stimulation of glucose uptake. Insulin exerts a number of different effects, driven by mechanisms that are only partially understood. In intracellular delivery of signals, certain kinases and phosphatases work together in a coordinated manner. Dissociation of insulin from the receptor is not sufficient to divert the signals induced by insulin. Tyrosine kinase activity of the insulin receptor lasts as long as the regulatory domain remains phosphorylated. Cellular PTPases lead to signal transduction. Pharmacologically active compounds that have an inhibitory effect on negative regulators of the insulin signaling pathway have a potential to delay dephosphorylation of the insulin receptor. This offers the possibility of using substances to reduce resistance to insulin.

Abbreviation

LAR leukocyte antigen-related protein-tyrosine-phosphatase

CD 45 leukocyte phosphatase CD 45

YOP Yersinia Protein-Tyrosine-Phosphatase

PTP α protein-tyrosine-phosphatase α

PTP 1B Protein-Tyrosine-Phosphatase 1B

TC-PTP T Cells-Protein-Tyrosine-Phosphatase

CDC-25 cell division regulation phosphatase 25

Phosphatase (Dual Specificity) in PTEN Chromosome 10

SHP1,2 src-homologous phosphatase 1,2

Example

1. Segmentation of DiFMUP According to Concentration of PTP 1B Enzyme

The reaction is carried out in black microtiter plates at a temperature of 37 ° C. 135 μl of reaction buffer per enzyme concentration to be assayed is provided, which comprises protein-tyrosine-phosphatase PTP 1B at the desired final concentration (FIG. 1: 30-600 ng / ml); 50 mM Hepes, pH 6.9; 150 mM NaCl; 1 mM EDTA; And 2 mM DTT components. The phosphatase reaction was initiated by adding 15 μl of 1 mM DiFMUP solution and the increase in fluorescence (measured by RFU) was measured for 15 minutes with a fluorescent microtiter plate photometer at excitation wavelength 358 nm and emission wavelength 455 nm. Measurement results of enzyme activity indicate that the fluorescence increases with the final concentration of PTP 1B, which can be shown graphically (FIG. 1).

2. Concentration dependence of DiFMUP cleavage by PTP 1B

The reaction is carried out in black microtiter plates at a temperature of 37 ° C. In each case, 135 μl of reaction buffer was provided, which was 100 ng protein-tyrosine-phosphatase PTP 1B / ml; 50 mM Hepes, pH 6.9; 150 mM NaCl; 1 mM EDTA; And 2 mM DTT components. The phosphatase reaction is initiated by adding 15 μl of DiFMUP solution, which comprises 10 times the substrate of the desired final concentration in the test mixture (FIG. 2: 0-200 μM), the fluorescence being 15 by fluorescence microtiter plate photometer at 358/455 nm. Measure at 30 second intervals for minutes. Measurement results of enzyme activity indicate that fluorescence (measured by RFU) increases with DiFMUP concentration, which can be shown graphically (FIG. 2). This graph can subsequently be used to determine the kinetic constant of the enzymatic reaction by Lineweaver-Burk analysis. Thus, PTP 1B was found to have a Km value of 19 μM and a Vmax of 388000 RF Usec ⁻¹ mg ⁻¹ . This assay can also be performed in a similar manner for other tyrosine phosphatase. Kinetic constants are listed in Table 1.

3. Segmentation of DiFMUP according to concentrations of phosphotyrosine phosphatase enzymes PTP α, LAR, T cell-PTP, SHP-2, CD 45 and YOP

The reaction is carried out in black microtiter plates at a temperature of 37 ° C. 135 μl of reaction buffer per enzyme and enzyme concentration to be assayed is provided, the reaction buffer being the desired final concentration (FIG. 3: PTP α: 0.5-1.85 μg / ml, LAR: 125-500 ng / ml; T cell-PTP : 66-330 ng / ml; CD 45: 50-400 ng / ml; YOP: 50-400 ng / ml; SHP-2: 0.3-2.4 μg / ml) protein-tyrosine-phosphatase; 50 mM Hepes, pH 6.9; 150 mM NaCl; 1 mM EDTA; And 2 mM DTT components. The phosphatase reaction was initiated by adding 15 μl of 1 mM DiFMUP solution and the fluorescence was measured at 15 seconds intervals for 15 minutes with a fluorescence microtiter plate photometer at 358/455 nm. The results of the measurement of enzyme activity indicate that the fluorescence (measured by RFU) increases with the final concentration of protein-tyrosine-phosphatase, which can be shown graphically (FIG. 3).

4. Measurement of Inhibitory Effect of PTP 1B on Phosphatase Inhibitors

Active compound 2,2-dioxo-2,3-dihydro-2,6-benzo [1,2,3] oxathiazol-5-yl)-(9-ethyl-9H-carbazole using DiFMUP Determination of inhibitory effect of -3-ylmethyl) amine The test is carried out on black microtiter plates at a temperature of 37 ° C. A 120 μl volume of reaction buffer was provided, which was 100 ng protein-tyrosine-phosphatase PTP 1B / ml; 50 mM Hepes, pH 6.9; 150 mM NaCl; 1 mM EDTA; And 2 mM DTT components. To this solution is added 15 μl of inhibitor solution to be tested for various purposes. The phosphatase reaction was initiated by adding 15 μl of 1 mM DiFMUP solution and the fluorescence (measured by RFU) was measured at 30 seconds apart for 15 minutes with a fluorescent microtiter plate photometer at 358/455 nm. Measurement results of enzyme activity indicate increased fluorescence, which can be shown graphically (FIG. 1). The reduction in enzyme activity obtained depends on the inhibitor concentration used. Active compound 2,2-dioxo-2,3-dihydro-2,6-benzo [1,2,3] oxathiazol-5-yl)-(9-ethyl-9H-carbazol-3-yl The inhibitor concentration at which methyl) amine reduces the activity of PTP 1B in half (1C-50) can be determined to be 3.8 μΜ. 1C-50 values using the pNPP assay and malachite green phosphopeptide test are measured for comparison. In this regard, the IC50 value is 5.1 μM by the pNPP assay and the IC50 value is 3.9 μM by the malachite green phosphopeptide test. The corresponding suppression curve is shown in FIG. 4.

5. Characterization of the type of inhibition exerted by phosphatase inhibitors on PTP 1B

The reaction is carried out in black microtiter plates at a temperature of 37 ° C. A 120 μl volume of reaction buffer was provided, which was 100 ng protein-tyrosine-phosphatase PTP 1B / ml; 50 mM Hepes, pH 6.9; 150 mM NaCl; 1 mM EDTA; And 2 mM DTT components and the inhibitor concentration depends on the previously determined IC50. The phosphatase reaction is initiated by adding 15 μl of DiFMUP solution, which comprises 10 times the substrate of the desired final concentration in the final volume (FIG. 2: 0 to 200 μm), the fluorescence being 358-455 nm until the reaction is saturated. Measured at 30 second intervals for 15 minutes with a fluorescent microtiter plate photometer at. Subsequently, more than 10 times the final concentration of the previously used substrate is added and the reaction is continuously monitored at 30 seconds intervals for 15 minutes with a fluorescence microtiter plate photometer at 358-455 nm. This reaction cannot be resumed in the presence of an irreversible inhibitor and can be resumed if the inhibition is of reversible type (FIG. 5).

6. Characterization by Time-Dependent Culture of Inhibition Type Exerted by Phosphatase Inhibitors on PTP 1B

The reaction is carried out in black microtiter plates at a temperature of 37 ° C. A 120 μl volume of reaction buffer was provided, which was 100 ng protein-tyrosine-phosphatase PTP 1B / ml; 50 mM Hepes, pH 6.9; 150 mM NaCl; 1 mM EDTA; And 2 mM DTT components and the inhibitor concentration depends on the previously determined IC50.

The mixture is incubated and at a given time point the phosphatase reaction is initiated by adding 15 μl of DiFMUP solution, which comprises a substrate 10 times the desired final concentration in the final volume (FIG. 2: 0 to 200 μm) and the fluorescence Measure at 30 seconds intervals for 15 minutes with a fluorescence microtiter plate photometer at 358-455 nm.

Reduction of enzyme activity in the presence of irreversible inhibitors depends on preculture time, while this phenomenon cannot be observed in the presence of inhibitors of the reversible inhibition type (FIG. 6).

List of Drawings:

1 shows the cleavage of DiFMUP depending on the concentration of PTP 1B enzyme.

2 shows the concentration dependence of DiFMUP cleavage by PTP 1B.

Figure 3 shows the cleavage of DiFMUP according to the concentration of phosphotyrosine phosphatase enzymes PTP α, LAR, TC-PTP, SHP-2, CD 45 and YOP.

Figure 4 shows the measurement of the inhibitory effect exerted by phosphatase inhibitors of PTP 1B.

5 shows the characterization of the type of inhibition of phosphatase inhibitors.

FIG. 6 shows characterization by time dependent culture of inhibition type of phosphatase inhibitors.

Claims (17)

Providing a biological material or an agent obtained from the biological material (a), (B) providing 6,8-difluoro-4-methylumbelliferylphosphate (DiFMUP), (C) contacting the biological material from step (a) or a preparation obtained from the biological material with DiFMUP from step (b) in an aqueous solution, and A method for detecting enzymatic activity of protein-tyrosine-phosphatase in a biological material, comprising the step (d) of detecting subsequently produced 6,8-difluoro-4-methylumbelliferyl by fluorometry. The method of claim 1, wherein at least one protein-tyrosine-phosphatase selected from the group LAR, CD 45, YOP, PTP α, PTP 1B, TC-PTP, CDC 25, PTEN, and SHP1,2 is provided as an agent obtained from a biological material. How to be. The method according to claim 1 or 2, wherein the concentration of DiFMUP after contacting is 10 to 250 µM. The method of claim 3 wherein the concentration of DiFMUP is between 50 and 100 μM. The process according to any one of claims 1 to 4, wherein the pH of the aqueous solution in step (c) is 5.0 to 8.0. The method of claim 5 wherein the pH is 6.0 to 7.5. The method of claim 5 or 8, wherein the pH is 7.0. Providing a chemical compound (a), (B) providing a biological material or an agent obtained from the biological material, (C) providing 6,8-difluoro-4-methylumbelliferylphosphate (DiFMUP), (D) contacting each other in an aqueous solution with the chemical compound from step (a), the biological material from step (b) or the agent obtained from the biological material and DiFMUP from step (c), (E) quantitatively determining the amount of 6,8-difluoro-4-methylumbelliferyl produced subsequently by fluorometry and Comparing the amount of 6,8-difluoro-4-methylumbelliferyl produced from step (e) with the amount of 6,8-difluoro-4-methylumbelliferyl produced by a control assay (f) A method for identifying a compound that changes the activity of protein-tyrosine-phosphatase, including. The method of claim 8, wherein the activity of protein-tyrosine-phosphatase is stimulated, inhibited or maintained. The method of claim 8 or 9, wherein the protein-tyrosine-phosphatase is selected from the groups LAR, CD 45, YOP, PTP α, PTP 1B, TC-PTP, CDC 25, PTEN and SHP1,2. A compound identified by the method according to claim 8. The compound of claim 11, wherein the mass is between 0.1 and 50 kDa. The compound of claim 11 or 12, wherein the mass is 0.1 to 5 kDa. The compound of any one of claims 11-13, wherein the mass is from 0.1 to 3 kDa. 15. The compound of any one of claims 11-14, which is a protein, amino acid, polysaccharide, sugar, polynucleotide, nucleotide, natural product or aromatic hydrocarbon compound. An auxiliary substance for formulating a medicament and / or a polymerizable additive, comprising a medicament comprising at least one compound according to any one of claims 11 to 15. Use of a compound according to any one of claims 11 to 14 for the manufacture of a medicament for the treatment of diabetes.