CN1612939A - Highly sensitive and continuous protein tyrosine phosphatase test using 6,8-difluoro-4-methylumbelliferyl phosphate - Google Patents

Highly sensitive and continuous protein tyrosine phosphatase test using 6,8-difluoro-4-methylumbelliferyl phosphate Download PDF

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CN1612939A
CN1612939A CNA028266668A CN02826666A CN1612939A CN 1612939 A CN1612939 A CN 1612939A CN A028266668 A CNA028266668 A CN A028266668A CN 02826666 A CN02826666 A CN 02826666A CN 1612939 A CN1612939 A CN 1612939A
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phosphatase
tyrosine
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S·韦尔特
N·特纳格尔斯
S·佩特里
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Sanofi Aventis Deutschland GmbH
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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

Use 6,8-two fluoro-4-methyl-umbelliferone phosphoric acid ester are extremely sensitive and detect the method for Protein-tyrosine-phosphatase (PTP enzyme) continuously
The present invention relates to use 6,8-two fluoro-4-methyl-umbelliferone phosphoric acid ester particularly detect improving one's methods of Protein-tyrosine-phosphatase under neutrallty condition.
Prior art discloses the use substrate, and right-nitrophenyl phosphoric acid ester (p-NPP) detects the active method of Protein-tyrosine-phosphatase.In standard biological chemistry handbook, can find these detection methods, as " Current Protocols in Protein Science:John E.Coligan (chief editor); Ben M.Dunn (chief editor); Hidde L.Ploegh (chief editor); David W.Speicher (chief editor), Paul T.Wingfield (chief editor); SBN:0-471-11184-8; Loose-leaf is brought in constant renewal in; John Wiley ﹠amp; Sons publishes ".The effect of Phosphoric acid esterase is to form right-nitrophenols from p-NPP.Detect right-nitrophenols based on the available light-intensity method of its strong yellow in alkaline range.
Yet this detection method has some unfavorable features.This detection is not suitable for directly determining the activity of this enzyme because it according to time-enforcement of terminations principle.Around this principle, add sodium hydroxide solution interruption enzyme reaction in the back in the past in given for some time.PH increase cause formed right-color of nitrophenols becomes yellow, determine its absorption and be the tolerance of right-nitrophenols amount with light-intensity method.It is quite detailed to enzyme dynamics that this detects principle, for example is used for determining to suppress type.Owing to need a large amount of experimental points, have to set up the independent experiment of respective numbers arranged side by side mutually.
In addition, substrate is to light, temperature and pH sensitivity.In the physiological pH scope, it has the trend of slow decomposition.When pNPP was used as substrate, some tyrosine phosphatases to be studied had maximum activity in acid range.For example, PTP1B demonstrates bigger conversion value at pH5.6.On the other hand, physiological pH 7.0 times, when using pNPP as substrate, PTP1B is only with 30% maximum conversion value work.This makes needs to use the enzyme of high relatively quantity, causes the corresponding increase of expense.Another unfavorable factor of this detection method is other composition that exists in detecting, and as damping fluid, salt or other material (detection material), absorption is arranged sometimes in yellow.This needs suitable background contrast.
The malachite green phospho-peptide detects and has been described as detecting the active another kind of method of Protein-tyrosine-phosphatase (Martin etc. (1985) Journal of Biological Chemistry, 260, (1994) Biochemical Journal such as 14932 pages and Harder, 298,395 pages).In the method, use malachite green reagent to detect the inorganic phosphate that Phosphoric acid esterase is discharged by its peptide substrate by light-intensity method.Except the wasting time and energy of the unstable of the photometry that causes owing to for example impurity or pH susceptibility and time-terminating method, another shortcoming is the specific substrates peptide of having to prepare each Phosphoric acid esterase of high density, and this makes this method quite expensive usually.
3,6-fluorescein bisphosphate has been described as detecting the another kind of substrate (Journal of Biomolecular Screening 4,327-334,1999) of Protein-tyrosine-phosphatase.Although this substrate makes it possible to directly measure enzymic activity with respect to above mentioned two substrates, the spectral quality of this substrate still is not good especially for implement measurement under the physiological pH scope.
Therefore, the present invention relates to the method for the enzymic activity of Protein-tyrosine-phosphatase in the detection of biological material, it comprises
A) goods that biologic material are provided or obtain from biologic material,
B) provide 6,8-two fluoro-4-methyl-umbelliferone phosphoric acid ester (DiFMUP),
C) in aqueous solution, make the goods that obtain from a) biologic material or from biologic material with from b) DiFMUP contact,
D) detect by fluorometric assay formed 6,8-two fluoro-4-methyl-umbelliferones.
The goods that obtain from biologic material preferably include and are selected from LAR, CD 45, PTP α, PTP1B, TCPTP, YOP, CDC 25, PTEN and SHP1,2 Protein-tyrosine-phosphatase.The goods that obtain from biologic material can exist with different purity grades.The goods that obtain from biologic material can be intact cell, decomposition cell, be rich in the sample of cellular constituent and/or organoid or purifying protein.
During when the contact biologic material or from goods that biologic material obtains, preferred 6, concentration from 10 to the 250 μ M of 8-two fluoro-4-methyl-umbelliferone phosphoric acid ester (DiFMUP).Preferred especially, the concentration of DiFMUP is from 50 to 100 μ M.
The pH that biologic material or the goods that obtain from biologic material contact with the aqueous solution of DiFMUP is preferably between 5.0 and 8.0, between 6.0 and 7.5.In one embodiment, preferred especially once more this pH is 7.0.
The present invention also relates to identify the method that changes the active compound of Protein-tyrosine-phosphatase, it comprises
A) provide a kind of compound,
B) goods that biologic material are provided or obtain from biologic material,
C) provide 6,8-two fluoro-4-methyl-umbelliferone phosphoric acid ester (DiFMUP),
D) in aqueous solution, make from a) compound with from b) biologic material or the goods that obtain from biologic material and from c) DiFMUP contact with each other,
E) determine 6 of formation by fluorometric assay, the amount of 8-two fluoro-4-methyl-umbelliferones,
F) form e relatively) 6, form in the amount of 8-two fluoro-4-methyl-umbelliferones and the controlled trial 6, the amount of 8-two fluoro-4-methyl-umbelliferones.
Controlled trial is characterised in that, during when biologic material or from goods contact DiFMUP that biologic material obtains, it is known for the effect of the Protein-tyrosine-phosphatase of selected type that no compound participates in above-mentioned step a) or this compound.Use in the controlled trial and its effect to Protein-tyrosine-phosphatase is that known described compound especially can be peptide or other compound of vanadate, vanadium organic compound, pervanadate, okadaic acid, NaF, dephostasin, modification.
In the various preferred embodiments of identifying the method that changes the active compound of Protein-tyrosine-phosphatase, this change should comprise the activity of stimulation, inhibition or stabilize proteins tyrosine phosphatase.
In another preferred embodiment of the method that identify to change the active compound of Protein-tyrosine-phosphatase, described Protein-tyrosine-phosphatase is selected from LAR, CD 45, PTP α, TC-PTP, CDC 25, PTEN, YOP, SHP1, and 2 and PTP1B.
The invention still further relates to and to change the Protein-tyrosine-phosphatase activity and carry out compounds identified with the method for the active compound of above-mentioned evaluation change Protein-tyrosine-phosphatase.Preferred this compound has 0.1 to 50kDa molecular weight, and more preferably 0.1 to 5kDa and more preferably 0.1 to 3kDa.This compound can be albumen, amino acid, polynucleotide, Nucleotide, natural product or arene compound.The invention still further relates to a kind of medicine, it comprises at least a aforesaid compound, and this compound is identified with the method for identifying the active compound of change Protein-tyrosine-phosphatase.This medicine also comprises the auxiliary substance and/or the polymeric additive of compounding pharmaceutical.
The invention still further relates to the method that is tested and appraised the active compound of change Protein-tyrosine-phosphatase and carried out of the application of the active compound of changed Protein-tyrosine-phosphatase of evaluation at the medicine of preparation treatment diabetes.
6,8-two fluoro-4-methyl-umbelliferone Phosphoric acid esterases can be from commercial acquisition.For example, European BV molecular probe company (2333 Leiden, Holland) sells this chemical.US 5,830, and 912 disclose its preparation.
Biologic material is any material that contains genetic information, and itself also can be bacterium or fungi such as intestinal bacteria or yeast saccharomyces cerevisiae.Biologic material also comprises the cell from cell cultures.
Under the situation of the cell of organizing from the animal or human, biologic material can take out or use syringe or conduit to take out or the similar techniques acquisition by examination of living tissue, operation.The cell of Qu Chuing can deep refrigeration, purifying or is cultivated like this.Use conventional microbial technique to make bacterium and yeast cell breeding and purifying.
Those skilled in the art can be at " Current Protocols in Molecular Biology; Chief editor: F.M.Ansabel etc., the loose-leaf publication is brought in constant renewal in, and 2001 editions, John Wiley ﹠amp; Sons publishes " the middle suitable guidance of finding for this purpose.Biologic material also can comprise the cell from animal cell culture.The example of this cell is mouse cell, rat cell or hamster cell.The cell of cell cultures can be the clone of primary cell type or foundation.The example of the clone of setting up is mouse 3T3 cell, Chinese hamster ovary celI or Hela cell.Standard textbook, for example " Basic Cell Culture; Chief editor: J.M.Daris IRL Press, Oxford (1996) " in keeping, grow and breeding of clone described.
By the goods of biologic material and purification step subsequently preparation that for example break from the biologic material acquisition.The method of biologic material of breaking especially can be multigelation, supersound process, use French squeezing machine or add washing composition and enzyme etc.Purification step subsequently for example comprises differential centrifugation, with ammonium sulfate or organic solvent deposit, use chromatographic technique etc.The example of chromatographic technique is polyacrylamide gel electrophoresis, high pressure liquid chromatography, ion-exchange chromatography, affinity chromatography, gas-chromatography, mass spectrum etc.For this purpose, those skilled in the art can utilize textbook as " Current Protocols inProtein Science, chief editor: J.E.Coligan etc., loose-leaf publication, continual renovation, 2001 editions, John Wiley ﹠amp; Sons publishes ", particularly about the detailed guidance of purifying protein.
Biologic material or the goods that obtain from biologic material can contact with DiFMUP the laboratory containers of routine such as Eppendorf pipe, centrifuge tube or glass flask.Water-containing medium under it contains for example buffer substance, nutritional medium component, single electric charge or double-charge ion such as Na +, K +, Ca 2+, Cl -, SO 4 2-And PO 3 2-, or other ion, and albumen, glycerine or another kind of material.For contact, specific controlled condition is as especially temperature, pH, ion condition, protein concentration, volume or other factors may be favourable.This is by for example in keeping the incubator device of steady temperature, in the presence of damping fluid or use the ion of accurately weighing in advance or albumen to implement to contact and finish.Water-containing solvent especially also can contain the organic solvent of specified proportion, as dimethyl sulfoxide (DMSO), methyl alcohol or ethanol.Yet the content of this solvent preferably accounts for 10% of volume of mixture at the most.
PTP enzyme (Phosphoric acid esterase) protein family comprises about 100 different members at present.These members can be subdivided into acceptor coupling protein and cytoplasm protein roughly.Phosphoric acid esterase has amino acid motif (H/V) CX jointly at catalyst structure domain 5R (S/T).Acceptor coupling Phosphoric acid esterase usually by extracellular domain, singlely stride the film district and one or two tenuigenin PTP enzymatic structure territory is formed.Think that LAR (white corpuscle has related antigen) albumen and PTP α albumen belong to acceptor coupling PTP enzyme.As for example result of " SH structural domain ", the PTP enzyme contains the various extensions in catalyst structure domain and C-terminal or N-terminal zone usually in the cell.These extend the function owing to target-seeking or adjusting.Enzyme PTP1B is classified as tenuigenin PTP enzyme.Except pure tyrosine phosphatase, the PTP enzyme family also comprises dual Phosphoric acid esterase group.Except Tyrosine O-phosphate, these enzymes also use phosphoserine or phosphothreonine as substrate.This group comprises for example Phosphoric acid esterase VHR and cdc25.
Phosphoric acid esterase LAR, PTP α, SHP-2 and PTP1B lead in the plain signal pathway that mediates at pancreas has critical function.These PTP enzymes are led the relevant and catalysis dephosphorylation reaction of plain acceptor with pancreas.These PTP enzymes may be alone or in combination in pancreas is led the pathogeny of plain resistance, work (Biochemistry38,3793-3803,1999; 3799 pages).
PTP1B is the negative conditioning agent that pancreas is led the plain signal transduction pathway that stimulates, and promptly this albumen cuts off once more by pancreas and leads plain inductive signal.By inference, this signal pathway is led the direct dephosphorylation of plain acceptor by pancreas and is interrupted.Also overexpression of PTP1B in a high proportion of patient who suffers from breast cancer.And this enzyme interacts with " Urogastron ".Verified this enzyme has two aromatic base phosphoric acid ester binding pockets.One just in time is positioned at the active centre, and another is positioned at position adjacent with catalytic center outside this.(Biochemistry?38，3793-3803，1999)。
In a lot of cells the transmission of signal and termination be subjected to comprise the factor tyrosine phosphorylation control.Complementary protein tyrosine kinase (PTK) and Phosphoric acid esterase (PTP enzyme), especially the active accurate balance of Protein-tyrosine-phosphatase has been set up phosphorylation state.
As enzyme, the PTP enzyme is responsible for making Tyrosine O-phosphate residue selectivity dephosphorylation.The PTP enzyme is being because for example somatomedin or hormone with the interaction of protein tyrosine kinase, in various biological procedures, function in conditioning signal.These signal transduction mechanisms play an important role in regulating cellular metabolism, growth, differentiation or mobility.
The fault of signal transduction pathway is regulated and is considered to one of reason of a lot of pathologic processes.These processes for example comprise cancer, some immunologys and neurological disease and also comprise type ii diabetes and obesity.
Compound provides by for example chemosynthesis.Those skilled in the art are familiar with the synthetic method of standard.This compound can be the part of compound set, stores and divides compounds and form from the synthesis program (being called chemical library) of having summed up.In other cases, this compound can be formed by microorganism, bacterium especially, or form (natural product) by fungi or plant.
The suitable drug compound of oral administration can exist with independent unit, as capsule, flat capsule, suction tablet or tablet, and as powder or granule, as solution in moisture or the on-aqueous liquid or suspension, or as oil-in-water or water-in-oil emulsion.These compositions can comprise the step that active compound is contacted with vehicle (it can be made up of one or more other components) according to any suitable pharmaceutical methods preparation.Usually, prepare said composition, if necessary, form product thereafter by active compound is mixed with liquid and/or finely divided solid excipient equably.
Therefore, tablet can be for example powder or particle by compacting or mold compound prepare, if wherein suitable, together with one or more other components.
The tablet of compacting can be by the compressing tablet free-flowing form compound such as powder or particle prepare, wherein in suitable machine suitably with tackiness agent, glidant, inert diluent and/or a kind of (several) surfactivity/dispersant.Can prepare the tablet of moulding by forming powder compound, it is got wet with inert liquid diluent in suitable machine.
The pharmaceutical composition that is fit to per os (hypogloeeis) administration comprises and contains compound and correctives, normally the lozenge of sucrose and gum arabic or tragacanth and in inert base such as gelatin and glycerine or sucrose and gum arabic the lozenge of inclusion compound.
The suitable drug composition of parenteral admin preferably includes the sterile aqueous preparations of compound, and said preparation is isoosmotic with the blood that is intended to the recipient preferably.Preferred intravenously gives these preparations, even also can be used as injection through subcutaneous, intramuscular or intradermal administration.Preferably can be by compound being mixed with water and making the solution of formation aseptic and ooze with blood etc. and to prepare these preparations.Usually the active compound that comprises 0.1 to 5% weight according to Injectable composition of the present invention.
The suitable drug composition of rectal administration preferably exists with the form of single dose suppository.
These can pass through compound and one or more conventional solid excipients, and for example theobroma oil mixes and the obtained by molding mixture prepares.
The local suitable drug composition that uses preferably exists with ointment, emulsifiable paste, lotion, paste, sprays, aerosol or finish form on the skin.Operable vehicle is the combination of Vaseline, lanolin, polyoxyethylene glycol, ethanol and two or more these materials.Active compound exists with 0.1 to 15% concentration of composition weight usually, and for example 0.5 to 2%.
Also can percutaneous dosing.The suitable drug composition that uses through skin can exist with the suitable form that contacts the independently plaster of patient's epidermis closely, for a long time.This plaster suitably comprises the active compound that is in the buffered aqueous solution, wherein is fit to, and dissolves and/or is scattered in the tackiness agent or be scattered in the polymkeric substance.Suitable activity compound concentration is about 1% to 35%, preferably approximately 3% to 15%.
The feature of diabetes B (the non-pancreas of NIDDM-is led plain dependent diabetes) be dextrose equivalent value height (hyperglycemia) under the fasting state (＞126mg/dl), lead plain resistance as the pancreas of muscle or fat in the peripheral tissues, the liver glyconeogenesis increases and pancreatic beta cell secretion pancreas is led plain not enough.The real causes of this disease is still unknown.Diabetes B often and other clinical condition resemble together and take place, as obesity, hypertriglyceridemia (rising of blood fat value) and hypertension.
By inference, to lead plain resistance be to understand the key of clinical condition elephant to pancreas.Pancreas is led plain resistance and is shown as the peripheral organ leads element to the pancreas of determining concentration response capacity reduction.This is reflected in cell levels, promptly induces pancreas to lead the plain required pancreas of effect and leads plain amount increase.In muscle, fat and liver cell, pancreas is led element glucose metabolism and metabolism of fat is had various effects, as increasing glucose from blood absorption, increases the accretion rate of glucose or suppresses the lipid acid decomposition.Think that development that various factors is led plain resistance at cell levels and pancreas has substantially and get in touch.Pancreas is led the factor of plain acceptor, signal cascade and the composition of glucose transport system plays an important role therein.
Pancreas is led element and is produced its biological action by lead plain acceptor in conjunction with pancreas in the first step.This receptor is led in conjunction with pancreas after the element, and its β subunit suffers pancreas to lead the autophosphorylation effect of plain receptor kinase.In muscle cell, this signal relies on IRS (pancreas is led plain receptor substrate) and PI3K (phosphoinositide-3-kinases) in the cell transmission and cause the glucose absorption that stimulated.Pancreas is led element and is brought a large amount of other effects, and these effects are undertaken by the mechanism of only part understanding.During signal transmitted in cell, specific kinases and Phosphoric acid esterase worked with coordination mode one.Pancreas is led plain dissociating from acceptor and is not enough to cut off pancreas and leads plain inductive signal.As long as the adjustment structure territory keeps phosphorylation, the tyrosine kinase activity that pancreas is led plain acceptor just keeps.Cell PTP enzyme is responsible for cutting off this signal.The inhibited pharmacologically active chemical compounds of negative conditioning agent that pancreas is led plain signal pathway has the pancreas of delay and leads the potentiality of plain acceptor dephosphorylation.This provides and can use reduction pancreas to be led the possibility of the material of plain resistance.
Abbreviation
The LAR LAR
The CD45 CD45
YOP yersinia Protein-tyrosine-phosphatase
PTP α Protein-tyrosine-phosphatase α
PTP 1B PTP 1B
TC-PTP T cell protein tyrosine Phosphoric acid esterase
CDC-25 cell fission control Phosphoric acid esterase 25
Phosphoric acid esterase (dual specificity) in the PTEN karyomit(e) 10
SHP1,2 src-homology phosphatase 1s, 2
Embodiment
1. depend on the cracking of the DiFMUP of PTP1B enzyme concn:
Be reflected under 37 ℃ and in the black microtiter plate, carry out.Every kind of enzyme concn to be analyzed provides 135 μ l reaction buffers, and described reaction buffer contains following ingredients: required final concentration (Fig. 1: Protein-tyrosine-phosphatase PTP1B 30-600ng/ml); 50mM Hepes, pH6.9; 150mMNaCl; 1mM EDTA; 2mM DTT.Add 15 μ l 1mM DiFMUP solution and begin phosphatase reaction, and (the unit: increase RFU), METHOD FOR CONTINUOUS DETERMINATION 15 minutes that in the fluorescence micro titer plate photometer, under excitation wavelength 358nm and emission wavelength 455nm, measures fluorescence.The tolerance of enzymic activity is that the fluorescence that depends on the PTP1B final concentration increases, and can illustrate description (Fig. 1).
2.PTP1B the concentration dependent of cracking DiFMUP
Be reflected under 37 ℃ and in the black microtiter plate, carry out.Every kind of situation provides 135 μ l reaction buffers, and described damping fluid contains following ingredients: 100ng Protein-tyrosine-phosphatase PTP1b/ml; 50mM Hepes, pH6.9; 150mM NaCl; 1mM EDTA; 2mM DTT.Add 15 μ lDiFMUP solution and begin phosphatase reaction, described solution contains substrate (Fig. 2: 0-200 μ M) of 10 times of required final concentrations in detecting mixture, and in the fluorescence micro titer plate photometer, under 358/455nm, measure fluorescence, with 30 seconds time interval determinations 15 minutes.The tolerance of enzymic activity is that (unit: increase RFU) can illustrate description (Fig. 2) to the fluorescence that depends on DiFMUP concentration.This figure can be used for analyzing to determine by Lineweaver-Burk the kinetic constant of enzyme reaction subsequently.Therefore, find that PTP1B has Km value and the 388000 RFU sec of 19 μ M -1Mg -1Vmax.This is analyzed also and can implement other tyrosine phosphatase with icotype.
3. depend on the cracking of the DiFMUP of phosphotyrosine phosphatase PTP α, LAR, T cell-PTP, SHP-2, CD45 and YOP concentration
Be reflected under 37 ℃ and in the black microtiter plate, carry out.Every kind of enzyme to be analyzed and enzyme concn provide 135 μ l reaction buffers, and described damping fluid contains following ingredients: the Protein-tyrosine-phosphatase of required final concentration (Fig. 3: PTP α: 0.5-1.85 μ g/ml, LAR:125-500ng/ml; T cell-PTP:66-330ng/ml; CD 45:50-400ng/ml; YOP:50-400ng/ml; SHP-2:0.3-2.4 μ g/ml); 50mM Hepes, pH6.9; 150mM NaCl; 1mM EDTA; 2mMDTT.Add 15 μ l 1mM DiFMUP solution and begin phosphatase reaction and in the fluorescence micro titer plate photometer, under 358/455nm, measure fluorescence, with 30 seconds time interval determinations 15 minutes.The tolerance of enzymic activity is the increase of the fluorescence (unit: RFU)) that depends on the Protein-tyrosine-phosphatase final concentration, can illustrate description (Fig. 3).
4. measure the restraining effect of the inhibitors of phosphatases of PTP1B
Use DiFMUP to detect and determine active compound 2,2-dioxo-2,3-dihydro-2,6-benzo [1,2,3] oxa-thiazole-5-yl)-the inhibiting detection of (9-ethyl-9H-carbazole-3-ylmethyl) amine carries out in the black microtiter plate under 37 ℃ of temperature.120 μ l reaction buffers are provided, and damping fluid contains following ingredients: 100ng Protein-tyrosine-phosphatase PTP1B/ml; 50mM Hepes, pH6.9; 150mM NaCl; 1mM EDTA; 2mM DTT.To the inhibitor solution to be detected that wherein adds the various concentration of 15 μ l.Add 15 μ l 1mM DiFMUP solution begin phosphatase reaction and in the fluorescence micro titer plate photometer, under 358/455nm, measure fluorescence (unit: RFU), with 30 seconds time interval determinations 15 minutes.The tolerance of enzymic activity is the increase of fluorescence, can illustrate description (Fig. 1).The enzymic activity that obtains reduces the inhibitor concentration that depends on employing.2,2-dioxo-2,3 dihydro-2,6-benzo [1,2,3] oxa-thiazole-5-yl)-(9-ethyl-9H-carbazole-3-ylmethyl) amine reduces active half the inhibitor concentration (IC-50) of PTP1B and can be defined as 3.8 μ M.Determine to use the IC-50 value of pNPP detection method and malachite green phospho-peptide detection method to compare.Thus, the pNPP detection method provides IC 50 values of 5.1 μ M and malachite green phospho-peptide detection method provides IC 50 values of 3.9 μ M.Fig. 4 comprises corresponding inhibition curve.
5. inhibitors of phosphatases is to the feature description of the inhibition type of PTP1b generation
Be reflected under 37 ℃ and in the black microtiter plate, carry out.The reaction buffer of 120 μ l volumes is provided, and damping fluid contains following ingredients: 100ng Protein-tyrosine-phosphatase PTP1b/ml; 50mMHepes, pH6.9; 150mM NaCl; 1mM EDTA; 2mM DTT and the inhibitor concentration that depends on the IC50 that determined in the past.Add 15 μ l DiFMUP solution and begin phosphatase reaction, described solution contains substrate (Fig. 2: 0-200 μ M) of 10 times of required final concentrations in final volume, and in the fluorescence micro titer plate photometer, under 358/455nm, measure fluorescence, with 30 seconds time interval determinations 15 minutes, reach capacity up to reaction.Subsequently, add the substrate of 10 times of final concentrations that are in excess in above employing and continue monitoring reaction, in the fluorescence micro titer plate photometer, under 358/455nm, measure fluorescence, with 30 seconds time interval determinations 15 minutes.When having irreversible inhibitor, can not begin reaction once more, but when suppressing to be reversible type, then be possible (Fig. 5).
6. hatch by time-dependent manner and describe the feature of inhibitors of phosphatases the inhibition type of PTP1b generation
Be reflected under 37 ℃ and in the black microtiter plate, carry out.120 μ l reaction buffers are provided, and damping fluid contains following ingredients: 100ng Protein-tyrosine-phosphatase PTP1b/ml; 50mM Hepes, pH6.9; 150mM NaCl; 1mM EDTA; 2mM DTT and the inhibitor concentration that depends on the IC50 that determined in the past.
Mixtures incubated is also added 15 μ l DiFMUP solution at the time point of determining and is begun phosphatase reaction, described solution contains substrate (Fig. 2: 0-200 μ M) of 10 times of required final concentrations in final volume, and in the fluorescence micro titer plate photometer, under 358/455nm, measure fluorescence, with 30 seconds time interval determinations 15 minutes.
In the presence of irreversible inhibitor, the reduction of enzymic activity depends on the time of preincubate, but and can not observe this phenomenon under the situation of the inhibitor of retroactive inhibition type.
Summary of drawings:
Fig. 1: depend on the cracking of the DiFMUP of PTP1B enzyme concn
Fig. 2: PTP1B is to DiFMUP cracked concentration dependent
Fig. 3: depend on the cracking of the DiFMUP of phosphotyrosine phosphatase PTP α, LAR, TCPTP, SHP2, CD45 and YOP concentration
Fig. 4: the restraining effect of measuring the inhibitors of phosphatases generation of PTP1B
Fig. 5: the evaluation of the inhibition type of inhibitors of phosphatases
Fig. 6: hatch the inhibition type of identifying inhibitors of phosphatases by time-dependent manner

Claims (17)

1. the method for the enzymic activity of Protein-tyrosine-phosphatase in the detection of biological material, it comprises
A) goods that biologic material are provided or obtain from biologic material,
B) provide 6,8-two fluoro-4-methyl-umbelliferone phosphoric acid ester (DiFMUP),
C) in aqueous solution, make the goods that obtain from a) biologic material or from biologic material with from b) DiFMUP contact,
D) detect 6 of formation, 8-two fluoro-4-methyl-umbelliferones by fluorometric assay.
2. method as claimed in claim 1, wherein, the Protein-tyrosine-phosphatase that at least a LAR of being selected from, CD 45, YOP, PTP α, PTP1B, TC-PTP, CDC 25, PTEN and SHP 1,2 are provided is as the goods that obtain from biologic material.
3. as the method for claim 1 or 2, wherein, the concentration of contact back DiFMUP is 10-250 μ M.
4. method as claimed in claim 3, wherein, the concentration of DiFMUP is from 50 to 100 μ M.
5. as one or multinomial described method of claim 1 to 4, wherein, c) pH of middle aqueous solution is between 5.0 and 8.0.
6. method as claimed in claim 5, wherein, pH is between 6.0 and 7.5.
7. as the method for claim 5 and 8, wherein pH is 7.0.
8. identify the method that changes the active compound of Protein-tyrosine-phosphatase for one kind, it comprises
A) provide a kind of compound,
B) goods that biologic material are provided or obtain from biologic material,
C) provide 6,8-two fluoro-4-methyl-umbelliferone phosphoric acid ester (DiFMUP),
D) in aqueous solution, make from a) compound with from b) biologic material or the goods that obtain from biologic material and from c) DiFMUP contact with each other,
E) determine 6 of formation by fluorometric assay, the amount of 8-two fluoro-4-methyl-umbelliferones,
F) form e relatively) 6, form in the amount of 8-two fluoro-4-methyl-umbelliferones and the controlled trial 6, the amount of 8-two fluoro-4-methyl-umbelliferones.
9. method as claimed in claim 8, the activity of wherein said Protein-tyrosine-phosphatase is stimulated, is suppressed or kept.
10. as the method for claim 8 or 9, Protein-tyrosine-phosphatase wherein is selected from LAR, CD 45, YOP, PTP α, PTP1B, TC-PTP, CDC 25, PTEN and SHP 1,2.
11. by having carried out compounds identified as the described method of claim 8 to 10.
12. as the compound of claim 11, wherein the molecular weight of compound 0.1 and 50KDa between.
13. as the compound of claim 11 or 12, wherein the molecular weight of compound 0.1 and 5KDa between.
14. as the compound of claim 11-13, wherein the molecular weight of compound 0.1 and 3KDa between.
15. as one or multinomial described compound of claim 11 to 14, wherein compound is albumen, amino acid, polysaccharide, sugar, polynucleotide, Nucleotide, natural product or arene compound.
16. a medicine comprises the auxiliary substance and/or the polymeric additive of the described at least a compound of claim 11 to 15, compounding pharmaceutical.
17. one of claim 11 to 14 or multinomial described compound are used for the treatment of application in the medicine of diabetes in preparation.
CNA028266668A 2002-01-04 2002-12-24 Highly sensitive and continuous protein tyrosine phosphatase test using 6,8-difluoro-4-methylumbelliferyl phosphate Pending CN1612939A (en)

Applications Claiming Priority (4)

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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
DE10200173.1 2002-01-04
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
DE10236329.3 2002-08-08

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CN113155787A (en) * 2020-12-29 2021-07-23 武汉合研生物医药科技有限公司 Activity detection method of mutant enzyme SHP 2E 76K

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CN100350045C (en) * 2003-08-21 2007-11-21 北京农业生物技术研究中心 Corn tyrosin protein phosphatase gene and its coding protein and use
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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