WO2009000965A1 - Homogeneous assay for kinase and phosphatase activity - Google Patents

Homogeneous assay for kinase and phosphatase activity Download PDF

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Publication number
WO2009000965A1
WO2009000965A1 PCT/FI2008/000080 FI2008000080W WO2009000965A1 WO 2009000965 A1 WO2009000965 A1 WO 2009000965A1 FI 2008000080 W FI2008000080 W FI 2008000080W WO 2009000965 A1 WO2009000965 A1 WO 2009000965A1
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peptide substrate
lanthanide
fluorescence
protein
chelate
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PCT/FI2008/000080
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French (fr)
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Yliopisto Turun
Pauliina NIEMELÄ
Tero Soukka
Heidi Appelblom
Timo Lövgren
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Yliopisto Turun
Niemelae Pauliina
Tero Soukka
Heidi Appelblom
Loevgren Timo
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Publication of WO2009000965A1 publication Critical patent/WO2009000965A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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

  • This Invention relates to fluorescent la ⁇ thanide chelate labelled peptide substrates for protein kinases and protein phosphatases and their uses in assays for protein kinases and protein phosphatases.
  • Protein phosphorylation is a common mechanism to selectively modify proteins carrying regulatory signalling in cells.
  • the enzymes that add phosphate groups to proteins are known as protein kinases.
  • the protein kinases come in two general classes, tyrosine kinases and serine/threonine kinases, which are defined by the amino acid that is phosphorylated.
  • Dephosphorylation is an opposite activity to protein phosphorylation and is carried out by another group of enzymes known as protein phosphatases. They can also be classified into two groups, tyrosine phosphatases and serine/threonine phosphatases, based on the amino acids whose dephosphorylation they catalyze.
  • the protein kinases and phosphatases are one of the largest and most attractive families of drug targets. About 30 % of human proteins contain covalently bound phosphate, and several hundred protein kinases and phosphatases are encoded by the humah genome. Thus, it is not surprising that abnormal phosphorylation i$ known to be a cause of major diseases, such as cancer and diabetes. Cohen, P. (2001) Eur. J. Biochem. 268: 5001-5010. In a drug discovery process, efficient screening of large molecular libraries demands that fast, simple, inexpensive and efficient high throughput screening methods are used.
  • Homogeneous assay format addition of reagents, mix and read, is the only way to perform high throughput screening of molecular libraries. It is essential that the assay is sensitive enough to detect positive hits and that also weak interactions are discovered. It is also important that the method is not prone to sample interference due to background fluorescence or autofluorescence.
  • fluorescence based assay systems cannot be monitored kinetically. They require sampling of the reaction mixture and typically the use of at least two separate labelling reactions in order to use a suitable label pair in FRET.
  • a simpler, kinetically monitorable fluorescence assay based on a one-label sensor detecting the phosphorylation status of a peptide that can be monitored real-time would obviously be the preferred method (Rothman, D.M. Trends Cell Biol. 2005: 15: 502-510).
  • Fluorophores for terminal labelling of peptide substrates and their use in measurement of protein kinase activity on the peptide substrates based on change of fluorescence intensity (Fl) of the fluorophore upon phosphorylation of the terminal amino acid are described in US 2005/0054024; by Yen, R-H. et al. (2002) J Biol Chem 277: 11527-11532 and Chen, C-A. (2004) Biochim Biophys Acta 1697: 39-51.
  • a fluorescent chemosensor resulting in change of fluorescence intensity of a sensing dye labelled peptide substrate upon phosphorylation of the peptide in presence of Mg 2+ has been described by Shults, M. D. (2005) Anal Biochem 352:198-207; Shults, M. D, et al. (2003) J. Am. Chem. Soc. 125:14284-14249, and Shults, M.D. et al. (2005) Nature Methods 2: 251 - 252.
  • Fluorescent lanthanide chelates that express a change in fluorescence intensity due to the environment and the use of these chelates in homogeneous immunoassays to measure analyte concentration based on luminescence modulation have been described by Hemmila, K et al. (1988) Clin Chem 34:2320- 23222; Mikola, H. et al. (1995), Bioconjugate Chem 6: 235-241; Barnard, G. et al. (1989) Clin Chem 35:555-559; and in EP 0324323.
  • Another object of the present invention is to provide a simplified, homogeneous time-resolved fluorescence based method for measurement of both protein kinase and protein phosphatase activity requiring only one labelled component and suitable for kinetic, real-time measurement of enzyme activity.
  • the present invention further provides uses of the methods of the invention to screen compounds for inhibition or activation of protein kinase or phosphatase activity.
  • Figure 1 illustrates the principle of a protein kinase and protein phosphatase activity assay based on phosphorylation sensitive enhancement or attenuation of the time-resolved fluorescence intensity of a lanthanide chelate labelled peptide substrate according to the invention upon phosphorylation or dephosphorylation, respectively.
  • Figure 2 illustrates the structure of the fluorescent terbium(llt) chelate, (2,2',2",2"V ⁇ 6,6'- ⁇ 4"-[2-(4-isothiocya ⁇ atophenyl)ethyl]pyrazole-1 Il ,3 lI -diyl ⁇ -bis(pyridi ⁇ e)-2,2 1 - diyl ⁇ bis(methylenenitrito) ⁇ tetrakis(acetato)terbium(lll)).
  • Figure 4, 5 and 6 illustrate results of protein phosphatase activity assays based on dephosphorylation sensitive attenuation of the time-resolved fluorescence intensity of a lanthanide chelate labelled phosphorylated peptide substrate according to the invention upon dephosphorylation due to phosphatase activity.
  • homogeneous assay method shall be understood to cover assays and assay methods requiring no separation steps. Single or multiple steps of each; addition of reagents, incubation and measurement are the only steps required.
  • fluorescence shall be understood to cover photoluminescence, i.e. luminescence excited by light, conventional short-lifetime fluorescence, delayed fluorescence with microsecond or millisecond fluorescence lifetime, tonic photoluminescence, and phosphorescence.
  • long-lifetime fluorescence and “long-lifetime fluorescent label” shall be understood to cover fluorescence and fluorescent labels having a luminescence lifetime equal to or more than 1 microsecond (the lifetime being calculated as the time wherein luminescence emission intensity decays to the relative value of 1/e ; i.e. to approximately 37 % of the original luminescence emission intensity).
  • the compounds capable of long-lifetime fluorescence include fluorescent lanthanide chelates.
  • short-lifetime fluorescence and “short-lifetime fluorescent compound” shall be understood to cover fluorescence and fluorescent compounds with a luminescence lifetime of less than 1 microsecond.
  • fluorescent lanthanide chelate shall be understood to include lanthanide chelates and lanthanide cryptate structures, comprising one or more lanthanide ions, a chelating ligand, the chelating ligand comprising a chromophoric moiety able to absorb excitation light and transfer the excited energy to the chelated lanthanide ion, and optionally at least one functional group enabling the coupling of the chelate.
  • the fluorescent lanthanide chelates emit long-lifetime fluorescence upon excitation.
  • the lanthanide can represent one single lanthanide element or a combination of several different lanthanide elements.
  • time-resolved fluorescence and "time-resolved fluorescence intensity” shall be understood to include measurement of long-lifetime emission of the fluorescent lanthanide chelates in such a way that the measurement of the emission is not measured at the same time when the excitation light is exposed to the fluorescent lanthanide chelate; typically at least a 10 microsecond delay is used to separate the excitation light pulse and measurement of the emitted light, i.e. emission is measured after the excitation light is switched off.
  • Time-resolved fluorescence measurement of emission can be carried out using one or multiple cycles, each comprising an excitation light pulse, a short delay (at least 10 microseconds) and an actual measurement (typically for tens or hundreds of microseconds).
  • the obtained signal can be a sum or an average of the measurement cycles.
  • phosphorylatable amino acid shall be understood to include amino acids that can be phosphorylated enzymatically, i.e. phosphate is added in enzymatic reaction. These amino acids comprise tyrosine, threonine and serine.
  • the change in fluorescence intensity observed can be up to 400 %, thus demonstrating excellent performance for a homogeneous, simple assay for kinase and phosphatase activity.
  • Previous fluorescent lanthanide chelate-based methods for measurement of kinase or phosphatase activity have been based on indirect labelling of peptides using either a labelled streptavidin conjugate and biotinylated substrate or a labelled antibody specific to a phosphorylated amino acid.
  • the observation of the phosphorylation or dephosphorylation has been based on binding of two labels and measurement of time-resolved fluorescence resonance energy transfer (TR-FRET).
  • TR-FRET time-resolved fluorescence resonance energy transfer
  • the present invention describes the discovery of a direct method to time-resolved fluorescence intensity reading based measurement of change of phosphorylation degree in the lanthanide chelate labelled peptide substrate. This allows elimination of the short-lifetime fluorescent background and autofluorescence, and use of lower peptide substrate concentrations than possible otherwise.
  • the present invention provides a simplified method for measurement of both protein kinase and protein phosphatase activity using time-resolved fluorometry, where the time-resolved fluorescence intensity of a fluorescent lanthanide chelate labelled substrate is dependent on the phosphorylation degree of the substrate.
  • the invention also provides kinetic measurement of protein kinase and protein phosphatase activity.
  • the fluorescent lanthanide chelate is attached to the peptide substrate at a distance shorter than twelve amino acids from one of the phosphorylated or phosphorylatable amino acids. In more preferred embodiments at least one distance in the peptide substrate is shorter than 9 amino acids and in most preferred embodiments it is shorter than 6 amino acids.
  • the lanthanide chelate is attached to the amino group of the lysine side chain and the phosphorylatable or phosphorylated amino acid is tyrosine or phosphotyrosine, respectively, referred to as "Y" in the sequence.
  • Protein tyrosine kinase can add a phosphate group to a tyrosine amino acid in the peptide sequence and an increase (+400 %) in time-resolved fluorescence intensity of the lanthanide chelate is observed.
  • protein tyrosine phosphatase can remove a phosphate group from a phosphotyrosine amino acid in the peptide sequence and a decrease (-80%) in time-resolved fluorescence intensity is observed.
  • time-resolved fluorescence intensity of the fluorescent lanthanide chelate is observed, but in some preferred embodiments.it is also possible to measure the decay constant of long-lifetime emission and observe a change in the decay constant.
  • the fluorescent lanthanide chelate is covalently coupled to the terminal amino group of the peptide substrate, to an amino group of the lysine side chain, to a thiol group of the cysteine side chain or to a suitable group in some artificial amino acid in the peptide substrate, or a specific building block is used during the synthesis of the peptide substrate to introduce the fluorescent lanthanide chelate as inherent part of the peptide substrate.
  • the phosphorylated and the nonphosphorylated amino acids are selected from tyrosine, threonine and serine and their phosphorylated counterparts i.e. phosphotyrosine, phosphothreonine and phosphoserine,
  • the lanthanide chelate is attached to the peptide substrate by a covalent bond. In some preferred embodiments the lanthanide chelate is attached to the peptide substrate by a non-covalent bond.
  • the fluorescent lanthanide chelate is preferably a fluorescent europium(lll) or terbium(lll) chelate, and most preferably a fluorescent terbium(III) chelate.
  • the fluorescent lanthanide preferably comprises a 2,2'-(1H-pyrazole-1 ,3- diyl)dipyridine chromophoric moiety.
  • the most preferred fluorescent lanthanide chelate is (2,2 l ,2",2 ni - ⁇ 6 > 6 1 - ⁇ 4 lt -I2-(4- isothiocyanatophenyl)ethyl]pyrazole-1",3"-diyl ⁇ -bis(pyridine)-2,2 t diyl ⁇ bis(methylene- nitrilo) ⁇ tetrakis(acetato)terbium(lll)).
  • the methods of the invention can be applied for many purposes. These methods can be used for screening compound for their efficacy to either inhibit or activate protein kinase activity and/or protein phosphatase activity. The methods are also very useful when determining the mechanism of action of any phenomenon involving protein kinase and/or protein phosphatase activity. The methods can be useful in drug screening as well as in screening of harmful effects of compounds.
  • the phosphorylatio ⁇ -sensitive lanthanide chelate (2,2',2",2'"-((6,6'-(4"-[2-(4-JsO- thiocyanatophenyl)ethyl]pyrazole-1",3"-diyl ⁇ -bis(pyridine)-2,2 l -diyl ⁇ bis(methylene'. ⁇ itrilo) ⁇ tetrakis(acetato)terbiurn(lll)) (Fig. 2) consists of an energy absorbing moiety, two chelating groups and a reactive group, which allows the chelate to be attached to the peptide. Chelate was synthesized as previously described (US 5,571,897) and stored at -20 ' C.
  • Chelate was diluted to 50 mmol/L carbonate buffer, pH 9.8, prior to use and the concentration of the chelate solution was determined by diluting the chelate to DELFIA enhancement solution (University of Turku, Finland) and using Tb 3+ -ion as standard.
  • DELFIA enhancement solution Universality of Turku, Finland
  • Monoclonal anti-phosphotyrosine antibody (1,6 mg/ml, Lot 10) was obtained from Cell Signaling Technologies (USA).
  • Antibody was biotinylated with biotin- isothiocyanate (University of Turku, Finland) in 50 mmol/L carbonate buffer, pH 9.8, with 50-fold molar excess of the biotin reagent. The reaction was incubated for 4 hours at room temperature after which free biotin was removed with NAP-5 and NAP-10 gel filtration columns (GE Healthcare Bio-sciences, Uppsala, Sweden) following the manufacturer's instructions using 50 mmol/L Tris- HCI pH 7.75 containing 0.9 % (w/v) NaCI and 0.05 % (w/v) NaN 3 . Biotinylated and purified antibody was further supplemented with 0.1 % BSA (bovine serum albumin) and stored at 4 C.
  • BSA bovine serum albumin
  • Lysine amino acids (K) of each peptide were labelled with the fluorescent terbium(lll) chelate.
  • the labelling reactions of the peptides were performed in 50 mmol/L carbonate buffer (pH 9,8) overnight at 4 ° C.
  • the concentration of the peptides was 3.3-6.3 mg/ml and a one to five -fold molar excess of the label over lysine residues were used.
  • the labelled peptides were purified with reverse-phase HPLC chromatography using a ⁇ RPC C2/C18-column (Amersham Bioscie ⁇ ces, Uppsala, Sweden) and an increasing MeCN gradient (from 5 % to 100 %). Fractions were collected based on monitoring absorbance at 280 ⁇ m.
  • the homogeneous assay for kinase activity was performed in Maxisorb microtitration wells (NUNC, Roskilde, Denmark) that were passively coated with BSA to prevent non-specific binding. All the assay components were diluted to 50 mmol/L Tris-HCI, pH 7.5, 10 mmol/L MgCI 2 , 1 mmol/L EGTA, 0.01 % (v/v) Brij35, ,2 mmol/L DTT, as suggested the optimal buffer for the enzyme. Different amounts of kinase, ATP and unphosphorylated peptide were tested and the reactions were performed in 10 or 50 ⁇ l volume.
  • Phosphatase assay was performed in BSA-coated wells (see above) and all dilutions were made to 25 mmol/L Tris-HCI ,pH 7.5, 100 mmol/L NaCI, 2 mmol/L Na 2 EDTA, 0.01 % (v/v) Brij 35, 5 mmol/L DTT, 1 mg/mt BSA. Different amounts of phosphatase (in 25 ⁇ l volume) and phosphoryiated peptide (in 25 ⁇ l volume) were tested. Reactions were incubated at 30 " C for one hour after which the terbium signal (545 nm) was measured with Victor 1420 Multilabel Reader.
  • kinase and phosphatase enzymes to phosphorylate and dephosphorylate peptide substrates was assured performing a heterogeneous assay using anti-phosphotyrosine antibody.
  • Biotinylated antibody 200 ng/well was attached to streptavidin coated wells (lnnotrac Diagnostics, Turku, Finland) after which the plates were incubated for 30 minutes at room temperature in low shaking.
  • Wells were washed twice using DELFIA wash buffer after which samples from the kinase or phosphatase reactions were transferred to the antibody-coated - wells. Reactions were incubated for one hour at room temperature in low shaking, washed twice, and the terbium signal was developed as described above.
  • Z' factor 1 - ((3SD of sample) + (3SD of control) /
  • Table 1 shows the results of time-resolved fluorescence intensity measurements using 50 ⁇ M ATP concentration, 5 nM peptide substrate (EAI-Y-AAPFAK-Tb(III), where fluorescent terbium(lll) chelate, referred to as Tb(III), is attached to an amino group of the lysine side chain) and 0-10 U of AbI tyrosine kinase in the reaction. The reaction was incubated overnight.
  • # S/B represents signal-to-background ratio calculated as ratio between signal of reaction with 0.5-10 U of kinase and signal of reaction without kinase.
  • a homogeneous protein tyrosine phosphatase activity experiment was carried out using 25 nM peptide substrate Tb(II l)-KEE-pY-EEEE-pY-EEEE-pY-EE (i.e. poly- GT-peptide substrate that comprises three internal phosphotyrosines, referred to as pY, and one fluorescent terbium(lll) chelate, referred as Tb(III), attached to the side chain of terminal lysine), variable amounts of enzyme (total reaction volume 25 ⁇ l_) and with different reaction times.
  • Results shown in Fig. 4 indicate that the phosphatase reaction is very rapid already at the smallest phosphatase concentration and all the substrate is dephosphorylated in 15 minutes.
  • Fig. 5 shows the effect of reduced substrate concentration (10, 5, 3, 1 and 0.5 nM) using a 15 min reaction time and 0.5 U enzyme/reaction; the obtained signal-to- background ratio stays constant with smaller substrate concentrations indicating that the method is also suitable for small substrate concentrations.
  • the effect of the distance between the chelate and phosphoryl group was studied by testing four other peptide substrates in the homogeneous protein tyrosine phosphatase assay.
  • the distance between the lanthanide and the phosphoryl group was from 3 to 6 amino acids; the employed peptide substrates were EAI-p ⁇ Y-.

Abstract

This invention relates to a peptide substrate comprising one or more phosphorylated and/or phosphorylatable amino acids and labelled with a fluorescent lanthanide chelate wherein the fluorescence of the lanthanide chelate changes depending on the degree of phosphorylation of the peptide substrate. This invention also relates to a method for determining protein kinase activity wherein the peptide substrate of the invention acts as a substrate of a protein Kinase or phosphatase and wherein the determination of said protein kinase or phosphatase activity is based on that the fluorescence of the lanthanide chelate changes when one or more phosphorous groups are added or removed, respectively, to said peptide substrate. The invention further relates to the use of the methods of the invention for screening of compounds for inhibition or activation of protein kinase activity or protein phosphatase activity.

Description

HOMOGENEOUS ASSAY FOR KINASE AND PHOSPHATASE ACTIVITY
FIELD OF THE INVENTION
This Invention relates to fluorescent laπthanide chelate labelled peptide substrates for protein kinases and protein phosphatases and their uses in assays for protein kinases and protein phosphatases.
BACKGROUND OF THE INVENTION
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details with respect to the practice, are incorporated by reference.
Protein phosphorylation is a common mechanism to selectively modify proteins carrying regulatory signalling in cells. The enzymes that add phosphate groups to proteins are known as protein kinases. The protein kinases come in two general classes, tyrosine kinases and serine/threonine kinases, which are defined by the amino acid that is phosphorylated.
Dephosphorylation is an opposite activity to protein phosphorylation and is carried out by another group of enzymes known as protein phosphatases. They can also be classified into two groups, tyrosine phosphatases and serine/threonine phosphatases, based on the amino acids whose dephosphorylation they catalyze.
Because of their important role in cellular signaling pathways, the protein kinases and phosphatases are one of the largest and most attractive families of drug targets. About 30 % of human proteins contain covalently bound phosphate, and several hundred protein kinases and phosphatases are encoded by the humah genome. Thus, it is not surprising that abnormal phosphorylation i$ known to be a cause of major diseases, such as cancer and diabetes. Cohen, P. (2001) Eur. J. Biochem. 268: 5001-5010. In a drug discovery process, efficient screening of large molecular libraries demands that fast, simple, inexpensive and efficient high throughput screening methods are used. Homogeneous assay format, addition of reagents, mix and read, is the only way to perform high throughput screening of molecular libraries. It is essential that the assay is sensitive enough to detect positive hits and that also weak interactions are discovered. It is also important that the method is not prone to sample interference due to background fluorescence or autofluorescence.
The use of generic assay technologies to screen new targets for protein kinases or phosphatases facilitates assay development and decreases the time needed for implementation of assays in robotic screening. For tyrosine kinases, several generic assay technology platforms are available. These technologies make use of high-affinity antibodies that discriminate between phosphorylated and non- phosphorylated tyrosines. Similar generic antibodies specific for phosphoserine or phosphothreonine are lacking since these antibodies are known to be sequence specific. Most of the currently used non-radioactive HTS (high-throughput- screeπing) methods for possible drug candidates affecting kinase/phosphatase activity apply antibodies to detect phosphorylated proteins or peptides and the detection modalities include fluorescence polarization (FP)1 fluorescence quenching (FQ)1 fluorescence resonance energy transfer (FRET) and time- resolved fluorescence resonance energy-transfer (TR-FRET) [Ishida, A. et al. (2007) Journal of Pharmacological Sciences 103; 5-11; EP1748079; Wu1 JJ. (2002) Methods in Molecular Biology 190: 65-85; and Pope, AJ. (1999) Journal of Biomolecular Screening, 4: 301-302]. Most of the fluorescence based assay systems cannot be monitored kinetically. They require sampling of the reaction mixture and typically the use of at least two separate labelling reactions in order to use a suitable label pair in FRET. A simpler, kinetically monitorable fluorescence assay based on a one-label sensor detecting the phosphorylation status of a peptide that can be monitored real-time would obviously be the preferred method (Rothman, D.M. Trends Cell Biol. 2005: 15: 502-510).
Homogeneous assay principles to measure phosphatase activity using a fluorophore labelled peptide substrate and quenching of phosphorylated substrate due to binding of a polycationic quencher labelled polymer or a super quencher conjugated to a metal chelate are described in WO 2003/024946 and WO 2005/060626, respectively.
Fluorophores for terminal labelling of peptide substrates and their use in measurement of protein kinase activity on the peptide substrates based on change of fluorescence intensity (Fl) of the fluorophore upon phosphorylation of the terminal amino acid are described in US 2005/0054024; by Yen, R-H. et al. (2002) J Biol Chem 277: 11527-11532 and Chen, C-A. (2004) Biochim Biophys Acta 1697: 39-51.
A fluorescent chemosensor resulting in change of fluorescence intensity of a sensing dye labelled peptide substrate upon phosphorylation of the peptide in presence of Mg2+ has been described by Shults, M. D. (2005) Anal Biochem 352:198-207; Shults, M. D, et al. (2003) J. Am. Chem. Soc. 125:14284-14249, and Shults, M.D. et al. (2005) Nature Methods 2: 251 - 252.
A method for measurement of phosphorylating or dephosphorylating activity of an enzyme on a protein substrate based on change of fluorescence intensity of fluorescence lifetime upon phosphorylation or dephosphorylation of the fluorophore labelled substrate has been described in US 2003/0228646.
A homogeneous assay for protein phosphatases based on fluorescence intensity change of fluorescent-labelled phosphopeptide due to dephosphorylation has been describe by Noble, J. E. et al. (2003) Anal Chem 75: 2042-2047.
Fluorescent lanthanide chelates that express a change in fluorescence intensity due to the environment and the use of these chelates in homogeneous immunoassays to measure analyte concentration based on luminescence modulation have been described by Hemmila, K et al. (1988) Clin Chem 34:2320- 23222; Mikola, H. et al. (1995), Bioconjugate Chem 6: 235-241; Barnard, G. et al. (1989) Clin Chem 35:555-559; and in EP 0324323.
Development of improved fluorescence lanthanide chelates and their labelling reagents as well as peptide building blocks containing fluorescent lanthanide chelates that would have marginal change in fluorescence due to change in environment have been described by Takalo, H. et al. (1994) Biocoπjug Chem. 5: 278-282; Nishioka, T. et al. (2006) lnorg Chem, 45:4088-4096; Hovinen, J, (2007) Bioconjug Chem. 18: 597-600; Jaakkola, L et al. (2006) J Pept Sci. 12: 199-205; Peuralahtϊ, J. et al. (2002) Bioconjug. Chem. 13: 870-875; and US Patents 6080839 and 7018851.
Synthesis of a highly fluorescent terbium(Ml) chelate (2,2',2"I21"-{{6,6I-{4"-[2-(4- isothiocyanatophenyl)ethyl]pyra2θle-1",3""diyl}-'bis(pyridine)-2,2'diyl}bis(methylene- nitrilo)}tetrakjs(acetato)terbium(lll)) has been described EP 0770610.
Methods for protein kinase activity measurement based on fluorescent lanthanide chelates and peptide substrates have been described by Zhang, W. X. (2005) Anal. Biochem. 343: 76-83,
Monitoring of a nucleic acid amplification reaction based on time-resolved fluorescence intensity change of a fluorescent terbium(lll) chelate labelled oligonucleotide probe has been described by Nurmi, J. et al. (2000) Nucleic Acids Res. 28: e28, ii-vi; US 7,371 ,544 and EP 1255863.
OBJECTAND SUMMARY OF THE INVENTION
One object of the present invention is to provide a peptide substrate for enabling a simplified homogeneous time-resolved fluorescence based method for measurement of both protein kinase and protein phosphatase activity.
Another object of the present invention is to provide a simplified, homogeneous time-resolved fluorescence based method for measurement of both protein kinase and protein phosphatase activity requiring only one labelled component and suitable for kinetic, real-time measurement of enzyme activity.
A further object of the present invention is to provide uses of the method of the invention.
The present invention provides a peptide substrate comprising one or more phosphorylated and/or phosphorylatable amino acids and labelled with one or more a fluorescent lanthanide chelate. Characteristic for the peptide substrate is that the fluorescence of the lanthanide chelate changes depending on the degree of phosphorylation of said peptide substrate.
The present invention also provides a method for determining both protein kinase activity and protein phosphatase activity wherein the peptide substrate of the invention acts as a substrate of the protein kinase or protein phosphatase and wherein the determination of said protein kinase or protein phosphatase activity is based on that the fluorescence of the lanthanide chelate changes when one or more phosphorous groups are either added to or removed from the peptide substrate.
The present invention further provides uses of the methods of the invention to screen compounds for inhibition or activation of protein kinase or phosphatase activity.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the principle of a protein kinase and protein phosphatase activity assay based on phosphorylation sensitive enhancement or attenuation of the time-resolved fluorescence intensity of a lanthanide chelate labelled peptide substrate according to the invention upon phosphorylation or dephosphorylation, respectively.
Figure 2 illustrates the structure of the fluorescent terbium(llt) chelate, (2,2',2",2"V {{6,6'-{4"-[2-(4-isothiocyaπatophenyl)ethyl]pyrazole-1Il,3lI-diyl}-bis(pyridiπe)-2,21- diyl}bis(methylenenitrito)}tetrakis(acetato)terbium(lll)).
Figure 3 illustrates results of a protein kinase activity assay based on phosphorylation sensitive enhancement of the time-resolved fluorescence intensity of a lanthanide chelate labelled non-fluorescent nonphosphorylated peptide substrate according to the invention upon phosphorylation due to kinase activity.
Figure 4, 5 and 6 illustrate results of protein phosphatase activity assays based on dephosphorylation sensitive attenuation of the time-resolved fluorescence intensity of a lanthanide chelate labelled phosphorylated peptide substrate according to the invention upon dephosphorylation due to phosphatase activity.
DETAILED DESCRIPTION OF THE INVENTION
Definitions The terms "homogeneous assay method", "homogeneous assay" and "homogeneous method" shall be understood to cover assays and assay methods requiring no separation steps. Single or multiple steps of each; addition of reagents, incubation and measurement are the only steps required. The term "separation step" shall be understood to be a step where a labelled assay reagent bound onto a solid-phase, such as for example a microparticle or a microtitration well, is separated and physically isolated from the unbound labelled assay reagent; for example a microtitration well is washed (liquid is taken out and, to improve the separation, additional liquid is added and the well emptied) resulting in separation of the solid-phase bound labelled assay reagent from the labelled assay reagent not bound onto the solid-phase.
The term "fluorescence" shall be understood to cover photoluminescence, i.e. luminescence excited by light, conventional short-lifetime fluorescence, delayed fluorescence with microsecond or millisecond fluorescence lifetime, tonic photoluminescence, and phosphorescence.
The term "lanthanide" shall be understood here to be equivalent to "rare earth metal ion" and to include single lanthanide elements and any combination of several different lanthanide elements from the following: samarium, europium, terbium, dysprosium, especially europium and terbium.
The term "long-lifetime fluorescence" and "long-lifetime fluorescent label" shall be understood to cover fluorescence and fluorescent labels having a luminescence lifetime equal to or more than 1 microsecond (the lifetime being calculated as the time wherein luminescence emission intensity decays to the relative value of 1/e; i.e. to approximately 37 % of the original luminescence emission intensity). The compounds capable of long-lifetime fluorescence include fluorescent lanthanide chelates.
The term "short-lifetime fluorescence" and "short-lifetime fluorescent compound" shall be understood to cover fluorescence and fluorescent compounds with a luminescence lifetime of less than 1 microsecond.
The terms "fluorescent lanthanide chelate" shall be understood to include lanthanide chelates and lanthanide cryptate structures, comprising one or more lanthanide ions, a chelating ligand, the chelating ligand comprising a chromophoric moiety able to absorb excitation light and transfer the excited energy to the chelated lanthanide ion, and optionally at least one functional group enabling the coupling of the chelate. The fluorescent lanthanide chelates emit long-lifetime fluorescence upon excitation. The lanthanide can represent one single lanthanide element or a combination of several different lanthanide elements.
The term "time-resolved fluorescence" and "time-resolved fluorescence intensity" shall be understood to include measurement of long-lifetime emission of the fluorescent lanthanide chelates in such a way that the measurement of the emission is not measured at the same time when the excitation light is exposed to the fluorescent lanthanide chelate; typically at least a 10 microsecond delay is used to separate the excitation light pulse and measurement of the emitted light, i.e. emission is measured after the excitation light is switched off. Time-resolved fluorescence measurement of emission can be carried out using one or multiple cycles, each comprising an excitation light pulse, a short delay (at least 10 microseconds) and an actual measurement (typically for tens or hundreds of microseconds). The obtained signal can be a sum or an average of the measurement cycles.
The term "peptide substrate" shall be understood to include amino acids and their oligomers and polymers, i.e. peptides, that can act as substrate for specific enzymes. The peptide substrates must contain an active site; i.e. kinase peptide substrates have to contain a nσn-phosphorylated phosphorylatable amino acid and phosphatase peptide substrates must contain at least one phosphorylated amino acid. The amino acid sequence of the peptide substrate, especially the sequence near the active site, has a role in determining the specificity and activity of the enzyme for the substrate.
The term "phosphorylatable amino acid" shall be understood to include amino acids that can be phosphorylated enzymatically, i.e. phosphate is added in enzymatic reaction. These amino acids comprise tyrosine, threonine and serine.
The term "phosphorylated amino acid" shall be understood to include phosphorylated forms of certain amino acids, i.e. phosphorylatable amino acids containing a phosphate group. These amino acids comprise phosphotyrosine, phosphothreonine and phosphoserine.
The term "protein kinase" and "kinase" shall be understood to include enzymes that can catalyze phosphorylation (addition of phosphate/phosphoryl group) of phosphorylatable amino acids in proteins and/or peptides. These enzymes comprise, but are not limited to, protein tyrosine kinases, serine kinases and threonine kinases.
The term "protein phosphatase" and "phosphatase" shall be understood to include enzymes that can catalyze dephosphorylation of phosphorylated amino acids (removal of the phosphoryl/phosphate group) in proteins and/or peptides. These enzymes comprise, but are not limited to, protein tyrosine phosphatases, serine phosphatases and threonine phosphatases.
Contribution to the state of the art
There have been reports of assay systems based on substrates that contain a fluorophore positioned near the site of phosphorylation. Phosphorylation induces change in the fluorescence properties of the fluorophore, but the reported changes have been quite modest and the measurement has been based on conventional, short-lifetime fluorescence, which is prone to background interference and autofluorescence. The present invention involves a similar approach that is based on the use of a phosphorylation sensitive lanthanide chelate which time-resolved fluorescence intensity changes upon phosphorylation or dephosphorylation of a near-by amino acid residue of a lanthanide chelate labelled peptide substrate.
The change in fluorescence intensity observed can be up to 400 %, thus demonstrating excellent performance for a homogeneous, simple assay for kinase and phosphatase activity. Previous fluorescent lanthanide chelate-based methods for measurement of kinase or phosphatase activity have been based on indirect labelling of peptides using either a labelled streptavidin conjugate and biotinylated substrate or a labelled antibody specific to a phosphorylated amino acid. The observation of the phosphorylation or dephosphorylation has been based on binding of two labels and measurement of time-resolved fluorescence resonance energy transfer (TR-FRET). These assays have required at least two labelled components: one of the labelled components has been labelled with a fluorescent lanthanide chelate and another with conventional fluorophore, typically short- lifetime fluorescent label.
Change in fluorescence intensity of conventional, short-lifetime fluorophores have been used previously to observe phosphorylation of peptide substrates, but these dyes require high concentrations of the substrate and the measurement is prone to interference by background fluorescence and autofluorescence.
The present invention describes the discovery of a direct method to time-resolved fluorescence intensity reading based measurement of change of phosphorylation degree in the lanthanide chelate labelled peptide substrate. This allows elimination of the short-lifetime fluorescent background and autofluorescence, and use of lower peptide substrate concentrations than possible otherwise.
Preferred embodiments of the invention The present invention provides a simplified method for measurement of both protein kinase and protein phosphatase activity using time-resolved fluorometry, where the time-resolved fluorescence intensity of a fluorescent lanthanide chelate labelled substrate is dependent on the phosphorylation degree of the substrate. The invention also provides kinetic measurement of protein kinase and protein phosphatase activity. In many preferred embodiments the fluorescent lanthanide chelate is attached to the peptide substrate at a distance shorter than twelve amino acids from one of the phosphorylated or phosphorylatable amino acids. In more preferred embodiments at least one distance in the peptide substrate is shorter than 9 amino acids and in most preferred embodiments it is shorter than 6 amino acids. According to one embodiment as shown in Fig. 1 , the lanthanide chelate, referred as "F", is attached to the amino group of the lysine side chain and the phosphorylatable or phosphorylated amino acid is tyrosine or phosphotyrosine, respectively, referred to as "Y" in the sequence. Protein tyrosine kinase can add a phosphate group to a tyrosine amino acid in the peptide sequence and an increase (+400 %) in time-resolved fluorescence intensity of the lanthanide chelate is observed. In an opposite reaction, protein tyrosine phosphatase can remove a phosphate group from a phosphotyrosine amino acid in the peptide sequence and a decrease (-80%) in time-resolved fluorescence intensity is observed.
In most preferred embodiments time-resolved fluorescence intensity of the fluorescent lanthanide chelate is observed, but in some preferred embodiments.it is also possible to measure the decay constant of long-lifetime emission and observe a change in the decay constant.
In many preferred embodiments the fluorescent lanthanide chelate is covalently coupled to the terminal amino group of the peptide substrate, to an amino group of the lysine side chain, to a thiol group of the cysteine side chain or to a suitable group in some artificial amino acid in the peptide substrate, or a specific building block is used during the synthesis of the peptide substrate to introduce the fluorescent lanthanide chelate as inherent part of the peptide substrate.
According to yet another preferred embodiment the time-resolved fluorescence intensity of the lanthanide chelate is increased due to phosphorylation of the lanthanide chelate-labelled non-phosphorylated peptide substrate (and decreased due to dephosphorylation), but it is also possible to obtain a decrease in the time- resolved fluorescence intensity of other fluorescent lanthanide chelates due to phosphorylation (and increase due to dephosphorylation). In preferred embodiments the phosphorylated and/or phosphorylatable amino acid or acids are independently selected from the group consisting of tyrosine, serine or threonine. The fluorescent lanthanide chelate labelled substrate contains at least one phosphorylated amino acid or at least one nonphosphorylated phosphorylatable amino acid. Preferably the phosphorylated and the nonphosphorylated amino acids are selected from tyrosine, threonine and serine and their phosphorylated counterparts i.e. phosphotyrosine, phosphothreonine and phosphoserine,
In many preferred embodiments the lanthanide chelate is attached to the peptide substrate by a covalent bond. In some preferred embodiments the lanthanide chelate is attached to the peptide substrate by a non-covalent bond.
In typical embodiments of the invention the lanthanide chelate comprises a lanthanide ion and a chelating ligand, the chelating ligand comprising a chromophoric moiety able to absorb excitation light and transfer the excited energy to the chelated lanthanide ion.
The fluorescent lanthanide chelate is preferably a fluorescent europium(lll) or terbium(lll) chelate, and most preferably a fluorescent terbium(III) chelate.
The fluorescent lanthanide preferably comprises a 2,2'-(1H-pyrazole-1 ,3- diyl)dipyridine chromophoric moiety.
The most preferred fluorescent lanthanide chelate is (2,2l,2",2ni-{{6>61-{4lt-I2-(4- isothiocyanatophenyl)ethyl]pyrazole-1",3"-diyl}-bis(pyridine)-2,2tdiyl}bis(methylene- nitrilo)}tetrakis(acetato)terbium(lll)).
In many preferred methods of the invention involving a protein kinase the protein kinase is selected from the group consisting of tyrosine kinases, threonine kinases and serine kinases. In many preferred methods of the invention involving a protein phosphatase the protein phosphatase is selected from the group consisting of tyrosine phosphatases, threonine phosphatases and serine phosphatases.
The methods of the invention can be applied for many purposes. These methods can be used for screening compound for their efficacy to either inhibit or activate protein kinase activity and/or protein phosphatase activity. The methods are also very useful when determining the mechanism of action of any phenomenon involving protein kinase and/or protein phosphatase activity. The methods can be useful in drug screening as well as in screening of harmful effects of compounds.
EXAMPLES
Materials and methods
Kinases and phosphatases
Tyrosine kinase AbI and T-cell protein Tyrosine phosphatase (TC PTP, human) were purchased from New England Biolabs.
Peptide substrates
Peptide sequence Glu-Ala-lle-Tyr-Ala-Ala-Pro-Phe-Ala-Lys was ordered both as phosphorylated (EAI-pY-AAPFAK) and unphosphorylated (EAIYAAPFAK) forms from JPT Peptide Technologies (Berlin, Germany). Peptide sequences EAI-pY- APK, EAl-pY-AAPK and EAI-pY-AAAPK were from JPT Peptide Technologies (Berlin, Germany) and KEE-pY-EEEE-pY-EEEE-pY-EE from NeoMPS (France). All the used peptides were of > 90 % purity determined with mass spectrometry.
Fluorescent terbiumd ID chelate
The phosphorylatioπ-sensitive lanthanide chelate (2,2',2",2'"-((6,6'-(4"-[2-(4-JsO- thiocyanatophenyl)ethyl]pyrazole-1",3"-diyl}-bis(pyridine)-2,2l-diyl}bis(methylene'. πitrilo)}tetrakis(acetato)terbiurn(lll)) (Fig. 2) consists of an energy absorbing moiety, two chelating groups and a reactive group, which allows the chelate to be attached to the peptide. Chelate was synthesized as previously described (US 5,571,897) and stored at -20 'C. Chelate was diluted to 50 mmol/L carbonate buffer, pH 9.8, prior to use and the concentration of the chelate solution was determined by diluting the chelate to DELFIA enhancement solution (University of Turku, Finland) and using Tb3+-ion as standard. Biotiπylated anti-phosphotyrosine antibody
Monoclonal anti-phosphotyrosine antibody (1,6 mg/ml, Lot 10) was obtained from Cell Signaling Technologies (USA). Antibody was biotinylated with biotin- isothiocyanate (University of Turku, Finland) in 50 mmol/L carbonate buffer, pH 9.8, with 50-fold molar excess of the biotin reagent. The reaction was incubated for 4 hours at room temperature after which free biotin was removed with NAP-5 and NAP-10 gel filtration columns (GE Healthcare Bio-sciences, Uppsala, Sweden) following the manufacturer's instructions using 50 mmol/L Tris- HCI pH 7.75 containing 0.9 % (w/v) NaCI and 0.05 % (w/v) NaN3. Biotinylated and purified antibody was further supplemented with 0.1 % BSA (bovine serum albumin) and stored at 4 C.
Labelling of peptides with fluorescent terbiumdlD chelate
Lysine amino acids (K) of each peptide were labelled with the fluorescent terbium(lll) chelate. The labelling reactions of the peptides were performed in 50 mmol/L carbonate buffer (pH 9,8) overnight at 4 °C. The concentration of the peptides was 3.3-6.3 mg/ml and a one to five -fold molar excess of the label over lysine residues were used. The labelled peptides were purified with reverse-phase HPLC chromatography using a μRPC C2/C18-column (Amersham Bioscieπces, Uppsala, Sweden) and an increasing MeCN gradient (from 5 % to 100 %). Fractions were collected based on monitoring absorbance at 280 πm.
Collected fractions were characterized further with biotinylated anti- phosphotyrosine antibody (150 ng/well) that had been attached to streptavidin coated wells (Innotrac Diagnostics, Turku, Finland). All the dilutions were made to assay buffer containing 50 mmol/L Tris-HCl, pH 7.75, 0.9 % (w/v) NaCI, 0.05.% (w/v) NaN3, 0.01 % (v/v) Tween 40, 0.05 % (w/v) bovine-γ-globulin, 20 μmol/L diethyleπetriaminepentaacetate and 0.5 % (w/v) BSA (Innotrac Diagnostics, Turku, Finland). After washes with DELFIA wash buffer (PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland), the signal was developed with the DELFIA enhancement solution and DELFIA enhancer (PerkinElmer Life and Analytical Sciences, Wallac Oy) and those fractions that gave signal were determined to contain the labelled peptide. The concentrations of the labelled peptides were then determined by measuring the Tb3+concentration against 1 πmol/L Tb3+ standard and the peptide concentration was determined by measuring absorbance at 280 nm. The absorbance of terbium at 280nm that affects the results was corrected in the calculations. Labelled peptides were stored at -204C in small aliquots.
Homogeneous assay for kinase activity
The homogeneous assay for kinase activity was performed in Maxisorb microtitration wells (NUNC, Roskilde, Denmark) that were passively coated with BSA to prevent non-specific binding. All the assay components were diluted to 50 mmol/L Tris-HCI, pH 7.5, 10 mmol/L MgCI2, 1 mmol/L EGTA, 0.01 % (v/v) Brij35, ,2 mmol/L DTT, as suggested the optimal buffer for the enzyme. Different amounts of kinase, ATP and unphosphorylated peptide were tested and the reactions were performed in 10 or 50 μl volume. Reactions were incubated at 30 °C for various time points after which the terbium signal (545 nm) was measured with Victor 1420 Multilabel Reader (Wallac, PerkinElmer Life and Analytical Sciences, Turku, Finland). The time-resolved fluorescence intensity measurement for terbium signal was carried out with a standard protocol for terbium, where the delay after UV excitation flash pulse (approximately 340 nm) was 500 μs and the measurement window time was 1400 μs. The measurement was typically repeated for 1000 cycles and the signal was represented as a sum of counted photons.
Homogeneous assay for phosphatase activity
Phosphatase assay was performed in BSA-coated wells (see above) and all dilutions were made to 25 mmol/L Tris-HCI ,pH 7.5, 100 mmol/L NaCI, 2 mmol/L Na2EDTA, 0.01 % (v/v) Brij 35, 5 mmol/L DTT, 1 mg/mt BSA. Different amounts of phosphatase (in 25 μl volume) and phosphoryiated peptide (in 25 μl volume) were tested. Reactions were incubated at 30 "C for one hour after which the terbium signal (545 nm) was measured with Victor 1420 Multilabel Reader.
Detection of phosphorylation/dephosphorylation with anti-P antibody
The functionality of the kinase and phosphatase enzymes to phosphorylate and dephosphorylate peptide substrates was assured performing a heterogeneous assay using anti-phosphotyrosine antibody. Biotinylated antibody (200 ng/well) was attached to streptavidin coated wells (lnnotrac Diagnostics, Turku, Finland) after which the plates were incubated for 30 minutes at room temperature in low shaking. Wells were washed twice using DELFIA wash buffer after which samples from the kinase or phosphatase reactions were transferred to the antibody-coated - wells. Reactions were incubated for one hour at room temperature in low shaking, washed twice, and the terbium signal was developed as described above.
Assay performance
To evaluate the quality of the kinase and phosphatase assays, Z' factor was calculated using equation : Z' = 1 - ((3SD of sample) + (3SD of control) / |mean of sample - mean of control! as described by Zhang, J.H. et al, (1999) J Biomol Screen. 4: 67-73,
Results
The effect of different amounts of kinase and phosphatase, ATP (in kinase reactions) and peptide substrate were tested and optimized in the homogeneous tyrosine kinase activity experiment. Table 1 shows the results of time-resolved fluorescence intensity measurements using 50 μM ATP concentration, 5 nM peptide substrate (EAI-Y-AAPFAK-Tb(III), where fluorescent terbium(lll) chelate, referred to as Tb(III), is attached to an amino group of the lysine side chain) and 0-10 U of AbI tyrosine kinase in the reaction. The reaction was incubated overnight. Signal-to-background ratio of 3.0 was achieved with 10 U of AbI Tyrosine kinase in reaction. Z1 calculated for 10 U reaction is 0.89 indicating significant difference between the measured signal levels and good performance of the assay method. Obtained results at different time points with varying ATP concentration are shown in Fig. 3. The kinase activity can be kinetically followed by measuring the time-resolved fluorescence intensity of the fluorescent terbium chelate at 545 nm.
The effect of staurosporine (known kinase inhibitor) on kinase activity was tested by adding different concentrations of the compound (0.1-50 μmol/L) to the reactions and observing the effect by measuring the time-resolved fluorescence intensity from reactions after the phosphorylation reaction. Table 1. Time-resolved fluorescence intensity readings from AbI tyrosine kinase ex eriment.
Figure imgf000018_0001
cv%* 4.6 % 1.8 % 2.1 % 0.3 % 1.0 %
S i BP 1.2 1.6 2.5 3.0
# S/B represents signal-to-background ratio calculated as ratio between signal of reaction with 0.5-10 U of kinase and signal of reaction without kinase.
$ CV% is coefficient of variation.
A homogeneous protein tyrosine phosphatase activity experiment was carried out using 25 nM peptide substrate Tb(II l)-KEE-pY-EEEE-pY-EEEE-pY-EE (i.e. poly- GT-peptide substrate that comprises three internal phosphotyrosines, referred to as pY, and one fluorescent terbium(lll) chelate, referred as Tb(III), attached to the side chain of terminal lysine), variable amounts of enzyme (total reaction volume 25 μl_) and with different reaction times. Results shown in Fig. 4 indicate that the phosphatase reaction is very rapid already at the smallest phosphatase concentration and all the substrate is dephosphorylated in 15 minutes. The distance between the lanthanide chelate and nearest tyrosine residue was 2 amino acids and the obtained signal-to-background ratio was over 4. Fig. 5 shows the effect of reduced substrate concentration (10, 5, 3, 1 and 0.5 nM) using a 15 min reaction time and 0.5 U enzyme/reaction; the obtained signal-to- background ratio stays constant with smaller substrate concentrations indicating that the method is also suitable for small substrate concentrations.
The effect of the distance between the chelate and phosphoryl group was studied by testing four other peptide substrates in the homogeneous protein tyrosine phosphatase assay. The distance between the lanthanide and the phosphoryl group was from 3 to 6 amino acids; the employed peptide substrates were EAI-p^Y-. APK-Tb(III), EAI-PY-AAPK-Tb(II I) and EAI-pY-AAAPK-Tb(lll). Results of the experiment are shown in Fig. 6. The highest signal-to-background ratio was obtained with the pσ!y-GT-peptide (S/B - 4) followed by the other peptide substrates so that the shorter the distance, the higher the sigπal-to-background ratio was achieved (varying from 3 to 2.7).
Other preferred embodiments
It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

Claims

1. A peptide substrate comprising one or more phosphorytated and/or phosphorylatable amino acids and labelled with a fluorescent lanthanide chelate characterized in that the fluorescence of said lanthanide chelate changes depending on the degree of phosphorylation of said peptide substrate.
2. The peptide substrate according to claim 1 , characterized in that the fluorescence is measured as time-resolved fluorescence.
3. The peptide substrate according to claim 1 or 2, characterized in that the phosphorylated and/or phosphorylatable amino acid or acids are independently selected from the group consisting of tyrosine, serine or threonine.
4. The peptide substrate according to claim 1, 2 or 3 characterized in that the lanthanide chelate is attached to said peptide substrate by a covalent bond.
5. The peptide substrate according to any of the preceding claims characterized in that the lanthanide chelate comprises a lanthanide ion and a chelating ligand, said chelating ligand comprising a chromophoric moiety able to absorb excitation light and transfer the excited energy to the chelated lanthanide ion.
6. The peptide substrate according to claim 5 characterized in that the lanthanide is terbium.
7. The peptide substrate according to claim 5 characterized in that the chromophoric moiety of the lanthanide chelate comprises a 2,2'-(1H-pyrazole-1 ,3- diyl)dipyridine moiety.
8. The peptide substrate according to claim 6 and 7 characterized in that said lanthanide chelates is (2,2',2",2'"-{{6,6'-{4""[2-(4-isothiocyanatophenyl)ethyl]- pyrazole-1",3"-diyl}-bis(pyridine)-2,2'-dϊyi}bis(methylenenitrilo)}tetrakis(acetato)- terbium(lll)).
9. A method for determining protein kinase activity wherein the peptide substrate of any of claims 1 to 8 acts as a substrate of said protein kinase and wherein the determination of said protein kinase activity is based on that the fluorescence of the lanthanide chelate changes when one or more phosphorous groups are added to said peptide substrate.
10. The method of claim 9 characterized in that the protein kinase is selected from the group consisting of tyrosine kinases, threonine kinases and serine kinases.
11. A method for determining protein phosphatase activity wherein the peptide substrate of any of claims 1 to 8 acts as a substrate of protein phosphatase and wherein the determination of protein phosphatase activity is based on that the fluorescence of the lanthanide chelate changes when one or more phosphorous groups are removed from said peptide substrate.
12. The method of claim 11 characterized in that the protein phosphatase is selected from the group consisting of tyrosine phosphatases, threonine phosphatases and serine phosphatases.
13. Use of the method of claim 9 or 10 in order to screen compounds for inhibition or activation of protein kinase activity.
14. Use of the method of claim 11 or 12 in order to screen compounds for inhibition or activation of protein phosphatase activity.
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