WO2001025477A2 - Kinase and phosphatase assays - Google Patents

Kinase and phosphatase assays Download PDF

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Publication number
WO2001025477A2
WO2001025477A2 PCT/GB2000/003736 GB0003736W WO0125477A2 WO 2001025477 A2 WO2001025477 A2 WO 2001025477A2 GB 0003736 W GB0003736 W GB 0003736W WO 0125477 A2 WO0125477 A2 WO 0125477A2
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Prior art keywords
substrate
reporter
kinase
phosphorylated
serine
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PCT/GB2000/003736
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French (fr)
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WO2001025477A3 (en
Inventor
Julie Ann Frearson
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Biofocus Discovery Limited
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Publication date
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Priority to AU75374/00A priority Critical patent/AU7537400A/en
Publication of WO2001025477A2 publication Critical patent/WO2001025477A2/en
Publication of WO2001025477A3 publication Critical patent/WO2001025477A3/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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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

Definitions

  • This invention relates to methods for assaying for kinase or phosphatase activity and to methods for screening for modulators of kinase or phosphatase activity, and to kits for use in methods according to the invention.
  • Protein-mteractmg modules help determine the specificity of signal transduction events, and protein phosphorylation can modulate the assembly of such modules into specific signalling complexes.
  • Kmases and phosphatases involved in regulating these assembly processes represent targets for possible therapeutic agents. Consequently, there is much interest in identifying kmases and phosphatases involved m signal transduction and in identifying modulators of kinase or phosphatase activity.
  • 14-3-3 proteins are a family of adaptor proteins which recognise and specifically associate with serine phosphorylated proteins (Muslin et al . , 1996 Cell 84, 889- 898). 14-3-3 proteins bind phosphoserme within the motif RSXXSpXP and therefore have the potential to associate with substrates of serine kmases. Two regions of 14-3-3 sequence have been proposed to contribute to phosphoserme binding: KNVIGAK at positions 50-57 and RY at positions 128 and 129. Both regions are highly conserved in a number of family members .
  • WW domains are small protein modules found in various proteins that participate in cell signalling. WW domain- containing proteins have been identified which function as phosphoserme or phosphothreonme binding modules (Lu et al . , 1999 Science 283, 1325-1382). An example is the peptidyl prolyl isomerase, Pm-l, which has a WW domain spanning residues 5-38 within its N-terminus. The sequence of this region is : EKLPPGWEKRMSRSSGRVY 23 YFNHITNASQW 34 ERPS . The residues critical for phosphoprotem binding have been identified as Tyr 23 and Trp 3 .
  • phosphoserme or phosphothreonme binding domains can be used in methods to screen for modulators of phosphoserme or phosphothreonme activity or for modulators of phosphatase activity.
  • phosphoserme or phosphothreonme binding domains can be used m methods to assay for phosphoserme or phosphothreonme kinase activity or for phosphatase activity.
  • a method for identifying a modulator of serine or threonine kinase activity which method is a screening assay which comprises: a) providing a serine or threonine kinase and a substrate which can be phosphorylated by the kinase; b) contacting the Kinase with its substrate m the presence and absence of a candidate modulator of the activity of the kinase under conditions which permit phosphorylation of the substrate by the kinase,• c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
  • a method for identifying a modulator of phosphatase activity which method is a screening assay which comprises: a) providing a substrate having a phosphorylated serine or threonine residue; b) contacting the substrate with a phosphatase the presence and absence of a candidate modulator of the activity of the phosphatase under conditions which permit dephosphorylation of the substrate by the phosphatase; c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
  • Methods according to the invention may further comprise identifying a modulator of serine or threonine kinase activity or of phosphatase activity.
  • the reporter may be any protein, other than a natural antibody (herein defined as an antibody raised m vivo against an antigen) , which binds a phosphorylated serine or threonine residue with higher affinity than the unphosphorylated residue.
  • a natural antibody herein defined as an antibody raised m vivo against an antigen
  • the reporter comprises a recombinant protein or a synthetic peptide.
  • the reporter may bind directly to a phosphorylated residue in the substrate.
  • the reporter binds to the phosphorylated residue substantially independently of the ammo acid sequence either side of the phosphorylated residue .
  • the reporter may be a 14-3-3 protein or a fragment or derivative thereof which retains phosphoserme residue binding activity.
  • the reporter may comprise the ammo acid residues of a 14-3-3 protein required for that protein to bind phosphoserme.
  • the reporter may comprise a protein having one or both of the ammo acid motifs KNVIGAKR and RY, which are highly conserved m 14-3-3 proteins.
  • the reporter may comprise a WW domain or a fragment or derivative thereof which retains phosphoserme residue or phosphothreonme residue binding activity.
  • the reporter may comprise a WW domain-containing protein such as Pm-l or a fragment or derivative thereof which retains phosphoserme or phosphothreonme residue binding activity.
  • the reporter may comprise a recombmant antibody or fragment or derivative thereof.
  • the reporter is not an antibody (whether natural or recombmant) or fragment or derivative thereof.
  • binding of the reporter to the substrate causes or allows a signal to be generated for measuring the binding of the reporter to the substrate.
  • the substrate may be immobilised to allow recordal of the signal.
  • Immobilisation of the substrate has been found to be particularly useful when methods of the invention are used for high throughput screening, especially if the substrate is immobilised in a flow cell or functionally equivalent apparatus which allows passage of liquid past the immobilised substrate.
  • the substrate may, for example be immobilised on magnetic particles, such as magnetic beads which are magnetically captured. Any immobilisation technique may be used. Suitable techniques which have been found to be particularly effective m methods of the invention include b ot ylat ng the substrate and immobilising the substrate by interaction with streptavidm, histidine tagging the substrate and immobilising the substrate by interaction with Nickel chelate, or fusing the substrate to a fusion domain which is immobilised by interaction with a natural antibody which recognises the fusion domain.
  • the signal generated may be a radioactive or a non- radioactive signal.
  • the signal generated is a non-radioactive signal when methods according to the invention are used for high throughput screening.
  • the signal generated may a luminescent signal, preferably an electrochemilummescent signal.
  • the reporter comprises a stable ruthenium metal chelate which generates electrochemilummescence in the presence of tnpropylamme when a voltage is applied to the stable ruthenium metal chelate .
  • kits for identifying a modulator of serine or threonine kinase activity which comprises a serine or threonine kinase, a substrate which can be phosphorylated by the kinase, and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate.
  • the kit may further comprise kinase buffer components.
  • the kit may further comprise one or more agents which are required for the reporter to generate a signal when it is bound to the substrate. One or more of the agents may be covalently linked to the reporter.
  • kits for identifying a modulator of phosphatase activity which comprises a substrate having a phosphorylated serine or threonine residue, a phosphatase which can dephosphorylate the serine or threonine residue of the substrate, and a reporter which binds phosphorylated substrate with higher affinity than unphosphorylated substrate.
  • the kit may further comprise phosphatase buffer components.
  • the kit may further comprise one or more agents which are required for the reporter to generate a signal when it is bound to the substrate. One or more of the agents may be covalently linked to the reporter.
  • a method for assaying for serine or threonine kinase activity which comprises: a) providing a substrate for a serine or threonine kinase ; b) contacting the substrate with a candidate serine or threonine kinase under conditions which permit phosphorylation of the substrate by a known serine or threonine kinase, • c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
  • kits for assaying for serine or threonine kinase activity which comprises a substrate which can be phosphorylated by a serine or threonine kinase and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate.
  • the kit may further comprise kinase buffer components .
  • the kit may further comprise one or more agents which are required for the reporter to generate a signal when it is bound to the substrate. One or more of the agents may be covalently linked to the reporter.
  • a method for assaying for phosphatase activity which comprises : a) providing a substrate having a phosphorylated serine or threonine res due; b) contacting the substrate with a candidate pnosphatase under conditions which permit dephosphorylation of the substrate by a known phosphatase; c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
  • kits for assaying for phosphatase activity which comprises a substrate having a phosphorylated serine or threonine residue and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate.
  • the kit may further comprise phosphatase buffer components.
  • the kit may further comprise one or more agents which are required for the reporter to generate a signal when it is bound to the substrate. One or more of the agents may be covalently linked to the reporter.
  • the reporter may be any protein other than a natural antibody which binds a phosphorylated serine or threonine residue with higher affinity than the unphosphorylated residue.
  • the reporter comprises a recombmant protein or a synthetic peptide.
  • Reporters excluding natural antibodies, which bind other phosphorylated residues, such as phosphotyrosme residues, may be used in methods to assay for kinase or phosphatase activity or for modulators of kinase or phosphatase activity in accordance with the invention.
  • a method for identifying a modulator of kinase activity which method is a screening assay which comprises: a) providing a kinase and a substrate which can be phosphorylated by the kinase ; b) contacting the kinase with its substrate in the presence and absence of a candidate modulator of the activity of the kinase under conditions which permit phosphorylation of the substrate by the kinase, • c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) m the absence of the candidate modulator.
  • a method for identifying a modulator of phosphatase activity which method is a screening assay which comprises: a) providing a substrate having a phosphorylated residue; b) contacting the substrate with a phosphatase in the presence and absence of a candidate modulator of the activity of the phosphatase under conditions which permit dephosphorylation of the substrate by the phosphatase; c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) m the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
  • the invention also provides a method for assaying for kinase activity which comprises: a) providing a substrate for a kinase ; b) contacting the substrate with a candidate kinase under conditions which permit phosphorylation of the substrate by a known kinase, c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
  • the invention also provides a method for assaying for phosphatase activity which comprises: a) providing a substrate having a phosphorylated residue,- b) contacting the substrate with a candidate phosphatase under conditions which permit dephosphorylation of the substrate by a known phosphatase; c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
  • Serine or threonine kinase was incubated in the presence and absence of a candidate modulator of kinase activity with a biotmylated peptide substrate in a kinase buffer
  • a WW domain-containing protein or 14-3-3 protein labelled with a stable ruthenium metal chelate (TAG) was added to each incubation and the incubations were then further incubated for 1 hour.
  • the stable ruthenium metal chelate generates electrochemilummscence (ECL) in the presence of tr propylamme upon voltage application.
  • ECL electrochemilummscence
  • the incubations were then channeled through a flow cell and the paramagnetic beads were captured at an electrode by magnetic application. Tripropylamme was added and voltage was applied. The resulting electrochemilummescence was measured using an Origen Analyser (Igen Inc.) .
  • Use of recombmant proteins or synthetic peptides n metnods according to the invention provide an economical, rapidly generated, non-exhaustible supply of reporter offering considerable practical advantages over antibodies.
  • the ECL technique described above has been found to be particularly appropriate to monitor binding of the reporter to the substrate in methods according to the invention because the large dynamic range, high signal to noise ratio and homogeneity of this technique provides high sensitivity and can be used for high throughput screening.
  • other methods for monitoring binding of the reporter to the substrate may be used.
  • other proximity assay-based techniques may be used, such as Homogeneous Time-Resolved Fluorescence (HTRF) .
  • HTRF Homogeneous Time-Resolved Fluorescence
  • the substrate may be tagged with polyhistidme and Ni 2* chelate bound deads may be used to capture the polyhistidme tagged substrate.
  • the substrate may be fused to a domain which can be specifically recognised by an antibody. Beads coated with the antibody may be used to capture the fusion domain fused to the substrate.
  • Glutathione S-Transferase GST
  • Methods according to the invention allow screening for serine or threonine kinase activity, or phosphatase activity and for modulators of such activities. Methods according to the invention are particularly advantageous for high througnput screening.

Abstract

Methods for screening for modulators of kinase or phosphatase activity are described. The methods comprise: a) providing a serine or threonine kinase and a substrate which can be phosphorylated by the kinase; b) contacting the kinase with its substrate in the presence and absence of a candidate modulator of the activity of the kinase under conditions which permit phosphorylation of the substrate by the kinase; c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator. Examples of reporters for use in such assays include 14-3-3 proteins and WW domain containing proteins.

Description

Assay
This invention relates to methods for assaying for kinase or phosphatase activity and to methods for screening for modulators of kinase or phosphatase activity, and to kits for use in methods according to the invention.
Protein-mteractmg modules help determine the specificity of signal transduction events, and protein phosphorylation can modulate the assembly of such modules into specific signalling complexes. Kmases and phosphatases involved in regulating these assembly processes represent targets for possible therapeutic agents. Consequently, there is much interest in identifying kmases and phosphatases involved m signal transduction and in identifying modulators of kinase or phosphatase activity.
Conventional in vi tro assays for kinase or phosphatase activity involve the determination of incorporation of radiolabelled phosphate into a substrate of the kinase or phosphatase. However, the quantities of radioactivity that are required to screen large chemical libraries for potential inhibitors of kinase or phosphatase activity can present problems because of the equipment and safety procedures required to deal with such levels of radioactivity. The rate of throughput of candidate inhibitors for assays which use radioactivity can also be lower than assays which do not use radioactivity because of limitations on the number of radioactive samples which can be analysed during high throughput screening by current equipment . The development of non-isotopic assays for screening large chemical libraries for modulators of kinase or phosphatase activity is therefore of considerable advantage. Antibodies which specifically recognise tyrosme phosphorylated residues are readily availabile and have been used in non- radioactive assays of tyrosme kinase activity and screening for inhibitors of tyrosme kmases. However, development of antibodies which specifically recognise phosphothreon e residues and, m particular, phosphoserme residues regardless of the surrounding ammo acid context has proved difficult. Polyclonal antibodies which recognise phosphothreonme and phosphoserme residues are available. However, some of these antibodies only recognise phosphoresidues in a particular sequence context and, therefore, will not bind to all phosphorylated serine or threonine kinase substrates. A further disadvantage is that a supply of polyclonal antibodies is not renewable once it has been exhausted. A different supply of polyclonal antibodies may have different phosphoresidue binding activity. This variation can cause inconsistencies the assay results . Antibodies are also t me consuming and difficult to produce and are, therefore, costly. This can be of great significance when large amounts of antibodies are used, for example when screening large libraries of chemical compounds .
Since many serine or threonine kmases represent excellent therapeutic targets, there is a need to provide assays for serine or threonine kinase activity and screening assays to identify modulators of these activities which are cost effective. In particular, it is desirable to provide high throughput screening assays to screen for modulators of serine or threonine kinase or phosphatase activity. 14-3-3 proteins are a family of adaptor proteins which recognise and specifically associate with serine phosphorylated proteins (Muslin et al . , 1996 Cell 84, 889- 898). 14-3-3 proteins bind phosphoserme within the motif RSXXSpXP and therefore have the potential to associate with substrates of serine kmases. Two regions of 14-3-3 sequence have been proposed to contribute to phosphoserme binding: KNVIGAK at positions 50-57 and RY at positions 128 and 129. Both regions are highly conserved in a number of family members .
WW domains are small protein modules found in various proteins that participate in cell signalling. WW domain- containing proteins have been identified which function as phosphoserme or phosphothreonme binding modules (Lu et al . , 1999 Science 283, 1325-1382). An example is the peptidyl prolyl isomerase, Pm-l, which has a WW domain spanning residues 5-38 within its N-terminus. The sequence of this region is : EKLPPGWEKRMSRSSGRVY23YFNHITNASQW34ERPS . The residues critical for phosphoprotem binding have been identified as Tyr23 and Trp3 . These residues together with Ser16 form a hydrophobic cluster within the WW domain and are likely to represent the phosphoserme or phosphothreonme binding pocket. Sequence alignment of WW domain-containing proteins indicates that these residues are highly conserved. This suggests that most WW domain proteins will bind phosphoserme or phosphothreonme residues.
We have appreciated that phosphoserme or phosphothreonme binding domains can be used in methods to screen for modulators of phosphoserme or phosphothreonme activity or for modulators of phosphatase activity. We have also appreciated that phosphoserme or phosphothreonme binding domains can be used m methods to assay for phosphoserme or phosphothreonme kinase activity or for phosphatase activity.
According to the invention there is provided a method for identifying a modulator of serine or threonine kinase activity, which method is a screening assay which comprises: a) providing a serine or threonine kinase and a substrate which can be phosphorylated by the kinase; b) contacting the Kinase with its substrate m the presence and absence of a candidate modulator of the activity of the kinase under conditions which permit phosphorylation of the substrate by the kinase,• c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
According to the invention there is also provided a method for identifying a modulator of phosphatase activity which method is a screening assay which comprises: a) providing a substrate having a phosphorylated serine or threonine residue; b) contacting the substrate with a phosphatase the presence and absence of a candidate modulator of the activity of the phosphatase under conditions which permit dephosphorylation of the substrate by the phosphatase; c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
Methods according to the invention may further comprise identifying a modulator of serine or threonine kinase activity or of phosphatase activity.
The reporter may be any protein, other than a natural antibody (herein defined as an antibody raised m vivo against an antigen) , which binds a phosphorylated serine or threonine residue with higher affinity than the unphosphorylated residue. Preferably the reporter comprises a recombinant protein or a synthetic peptide.
The reporter may bind directly to a phosphorylated residue in the substrate. Preferably the reporter binds to the phosphorylated residue substantially independently of the ammo acid sequence either side of the phosphorylated residue .
The reporter may be a 14-3-3 protein or a fragment or derivative thereof which retains phosphoserme residue binding activity. The reporter may comprise the ammo acid residues of a 14-3-3 protein required for that protein to bind phosphoserme. The reporter may comprise a protein having one or both of the ammo acid motifs KNVIGAKR and RY, which are highly conserved m 14-3-3 proteins.
The reporter may comprise a WW domain or a fragment or derivative thereof which retains phosphoserme residue or phosphothreonme residue binding activity. The reporter may comprise a WW domain-containing protein such as Pm-l or a fragment or derivative thereof which retains phosphoserme or phosphothreonme residue binding activity.
The reporter may comprise a recombmant antibody or fragment or derivative thereof. Preferably, however, the reporter is not an antibody (whether natural or recombmant) or fragment or derivative thereof.
Preferably binding of the reporter to the substrate causes or allows a signal to be generated for measuring the binding of the reporter to the substrate. The substrate may be immobilised to allow recordal of the signal.
Immobilisation of the substrate has been found to be particularly useful when methods of the invention are used for high throughput screening, especially if the substrate is immobilised in a flow cell or functionally equivalent apparatus which allows passage of liquid past the immobilised substrate.
The substrate may, for example be immobilised on magnetic particles, such as magnetic beads which are magnetically captured. Any immobilisation technique may be used. Suitable techniques which have been found to be particularly effective m methods of the invention include b ot ylat ng the substrate and immobilising the substrate by interaction with streptavidm, histidine tagging the substrate and immobilising the substrate by interaction with Nickel chelate, or fusing the substrate to a fusion domain which is immobilised by interaction with a natural antibody which recognises the fusion domain.
The signal generated may be a radioactive or a non- radioactive signal. Preferably the signal generated is a non-radioactive signal when methods according to the invention are used for high throughput screening.
The signal generated may a luminescent signal, preferably an electrochemilummescent signal. Preferably the reporter comprises a stable ruthenium metal chelate which generates electrochemilummescence in the presence of tnpropylamme when a voltage is applied to the stable ruthenium metal chelate .
Also provided according to the invention is a kit for identifying a modulator of serine or threonine kinase activity which comprises a serine or threonine kinase, a substrate which can be phosphorylated by the kinase, and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate. The kit may further comprise kinase buffer components. The kit may further comprise one or more agents which are required for the reporter to generate a signal when it is bound to the substrate. One or more of the agents may be covalently linked to the reporter.
Also provided according to the invention is a kit for identifying a modulator of phosphatase activity which comprises a substrate having a phosphorylated serine or threonine residue, a phosphatase which can dephosphorylate the serine or threonine residue of the substrate, and a reporter which binds phosphorylated substrate with higher affinity than unphosphorylated substrate. The kit may further comprise phosphatase buffer components. The kit may further comprise one or more agents which are required for the reporter to generate a signal when it is bound to the substrate. One or more of the agents may be covalently linked to the reporter.
According to the invention there is further provided a method for assaying for serine or threonine kinase activity which comprises: a) providing a substrate for a serine or threonine kinase ; b) contacting the substrate with a candidate serine or threonine kinase under conditions which permit phosphorylation of the substrate by a known serine or threonine kinase,• c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
Also provided according to the invention is a kit for assaying for serine or threonine kinase activity which comprises a substrate which can be phosphorylated by a serine or threonine kinase and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate. The kit may further comprise kinase buffer components . The kit may further comprise one or more agents which are required for the reporter to generate a signal when it is bound to the substrate. One or more of the agents may be covalently linked to the reporter.
There is further provided according to the invention a method for assaying for phosphatase activity which comprises : a) providing a substrate having a phosphorylated serine or threonine res due; b) contacting the substrate with a candidate pnosphatase under conditions which permit dephosphorylation of the substrate by a known phosphatase; c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
Also provided according to the invention s a kit for assaying for phosphatase activity which comprises a substrate having a phosphorylated serine or threonine residue and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate. The kit may further comprise phosphatase buffer components. The kit may further comprise one or more agents which are required for the reporter to generate a signal when it is bound to the substrate. One or more of the agents may be covalently linked to the reporter.
The reporter may be any protein other than a natural antibody which binds a phosphorylated serine or threonine residue with higher affinity than the unphosphorylated residue. Preferably the reporter comprises a recombmant protein or a synthetic peptide.
Reporters, excluding natural antibodies, which bind other phosphorylated residues, such as phosphotyrosme residues, may be used in methods to assay for kinase or phosphatase activity or for modulators of kinase or phosphatase activity in accordance with the invention.
Consequently, there is also provided according to the invention a method for identifying a modulator of kinase activity, which method is a screening assay which comprises: a) providing a kinase and a substrate which can be phosphorylated by the kinase ; b) contacting the kinase with its substrate in the presence and absence of a candidate modulator of the activity of the kinase under conditions which permit phosphorylation of the substrate by the kinase,• c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) m the absence of the candidate modulator.
There is further provided according to the invention a method for identifying a modulator of phosphatase activity which method is a screening assay which comprises: a) providing a substrate having a phosphorylated residue; b) contacting the substrate with a phosphatase in the presence and absence of a candidate modulator of the activity of the phosphatase under conditions which permit dephosphorylation of the substrate by the phosphatase; c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) m the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
The invention also provides a method for assaying for kinase activity which comprises: a) providing a substrate for a kinase ; b) contacting the substrate with a candidate kinase under conditions which permit phosphorylation of the substrate by a known kinase, c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
The invention also provides a method for assaying for phosphatase activity which comprises: a) providing a substrate having a phosphorylated residue,- b) contacting the substrate with a candidate phosphatase under conditions which permit dephosphorylation of the substrate by a known phosphatase; c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
An embodiment of the invention will now be described by way of example only.
Example
Serine or threonine kinase was incubated in the presence and absence of a candidate modulator of kinase activity with a biotmylated peptide substrate in a kinase buffer
(comprising 8mM MOPS pH 7, 0.2mM EDTA, lOmM MgCH3COO and 50μM
ATP) at room temperature for 10 minutes. The reaction was terminated by addition of 2 volumes of cold phosphate- buffered saline (pH 7.4). Streptavidm-coated paramagnetic beads were then added to the incubations which were then mixed with agitation for 30 minutes at room temperature.
A WW domain-containing protein or 14-3-3 protein labelled with a stable ruthenium metal chelate (TAG) was added to each incubation and the incubations were then further incubated for 1 hour. The stable ruthenium metal chelate generates electrochemilummscence (ECL) in the presence of tr propylamme upon voltage application. The incubations were then channeled through a flow cell and the paramagnetic beads were captured at an electrode by magnetic application. Tripropylamme was added and voltage was applied. The resulting electrochemilummescence was measured using an Origen Analyser (Igen Inc.) . Use of recombmant proteins or synthetic peptides n metnods according to the invention provide an economical, rapidly generated, non-exhaustible supply of reporter offering considerable practical advantages over antibodies.
The ECL technique described above has been found to be particularly appropriate to monitor binding of the reporter to the substrate in methods according to the invention because the large dynamic range, high signal to noise ratio and homogeneity of this technique provides high sensitivity and can be used for high throughput screening. However, t will be appreciated that other methods for monitoring binding of the reporter to the substrate may be used. For example, other proximity assay-based techniques may be used, such as Homogeneous Time-Resolved Fluorescence (HTRF) .
It will also be appreciated that alternative ways to immobilise the substrate may be used. For example, the substrate may be tagged with polyhistidme and Ni2* chelate bound deads may be used to capture the polyhistidme tagged substrate. Alternatively, the substrate may be fused to a domain which can be specifically recognised by an antibody. Beads coated with the antibody may be used to capture the fusion domain fused to the substrate. An example of a fusion domain is Glutathione S-Transferase (GST) .
Methods according to the invention allow screening for serine or threonine kinase activity, or phosphatase activity and for modulators of such activities. Methods according to the invention are particularly advantageous for high througnput screening.

Claims

Claims
1. A method for identifying a modulator of serine or threonine kinase activity, which method is a screening assay which comprises : a) providing a serine or threonine kinase and a substrate which can be phosphorylated by the kinase; b) contacting the kinase with its substrate in the presence and absence of a candidate modulator of the activity of the kinase under conditions which permit phospnorylation of the substrate by the kinase; c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
2. A method according to claim 1 further comprising identifying a modulator of serine or threonine kinase activity.
3. A method for identifying a modulator of phosphatase activity which method is a screening assay which comprises: a) providing a substrate having a phosphorylated serine or threonine residue,- b) contacting the substrate with a phosphatase in the presence and absence of a candidate modulator of the activity of the phosphatase under conditions which permit dephosphorylation of the substrate by the phosphatase; c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) m the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
4 A method according to claim 3 further comprising identifying a modulator of phosphatase activity.
5. A method according to any preceding claim m which the reporter is a recombmant protein or a synthetic peptide.
6. A method according to any preceding claim in which the reporter binds directly to a phosphorylated residue m the substrate .
7. A method according to claim 6 m which the reporter binds to the phosphorylated residue substantially independently of the ammo acid sequence either side of the phosphorylated residue.
8. A method according to any preceding claim in which the reporter comprises a 14-3-3 protein or a fragment or derivative thereof which retains phosphoserme residue binding activity.
9. A method according to any preceding m which the reoorter comprises the ammo acid residues of a 14-3-3 protein required for that protein to bind a phosphoserme residue .
10 A method according to any preceding claim m which the reporter comprises a protein having one or both of the ammo acid motifs KNVIGAKR and RY, which are highly conserved in 14-3-3 proteins.
11. A method according to any preceding claim in which the reporter comprises a WW domain or a fragment or derivative thereof which retains phosphoserme residue or phosphothreonme residue binding activity.
12. A method according to any preceding claim in which the reporter comprises a WW domain-containing protein or a fragment or derivative thereof .
13 A method according to any preceding claim m which binding of the reporter to the substrate causes or allows a signal to be generated for measuring the binding of the reporter to the substrate.
14 A method according to any preceding claim which further comprises immobilising the substrate to allow recordal of the signal.
15 A method according to claim 14 in which the substrate is immobilised in a flow cell
16 A method according to claim 14 or 15 m which the substrate is immobilised on magnetic particles, such as magnetic beads, and which the magnetic particles are captured magnetically
17. A method according to any of claims 14 to 16 in which the substrate is biotmylated and is immobilised by interaction with streptavidm.
18. A method according to any of claims 14 to 16 in which the substrate is histidme tagged and is immobilised by interaction with Nickel chelate.
19. A method according to any of claims 14 to 16 in which the substrate is fused to a fusion domain and is immobilised by interaction with an antibody which recognises the fusion domain.
20. A method according to any of claims 13 to 19 in which the signal generated is a non-radioactive signal.
21. A method according to claim 20 in which the signal generated is a luminescent signal.
22. A method according to claim 20 m which the signal generated is an electrochemilummescent signal.
23. A method according to claim 22 in which the reporter comprises a stable ruthenium metal chelate which generates electrochemilum escence in the presence of tripropylamme when a voltage is applied to the stable ruthenium metal chelate .
24. Use of a method according to any preceding claim for identifying a modulator of serine or threonine kinase activity .
25. A modulator of serine or threonine kinase activity identified using a method according to any of claims 1 to 23.
26. A screening assay for identifying a modulator of serine or threonine kinase activity which comprises the steps of claim 1.
27. A screening assay for identifying a modulator of phosphatase activity which comprises the steps of claim 3.
28. A kit for identifying a modulator of serine or threonine kinase activity which comprises a serine or threonine kinase, a substrate which can be phosphorylated by the kinase, and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate.
29. A kit for identifying a modulator of phosphatase activity which comprises a substrate having a phosphorylated serine or threonine residue, a phosphatase which can dephosphorylate the serine or threonine residue of the substrate, and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate .
30. A method for assaying for serine or threonine kinase activity which comprises: a) providing a substrate for a serine or threonine kinase,• b) contacting the substrate with a candidate serine or threonine kinase under conditions which permit phosphorylation of the substrate by a known serine or threonine kinase; c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
31. A method for assaying for phosphatase activity which comprises : a) providing a substrate having a phosphorylated serine or threonine residue; b) contacting the substrate with a candidate phosphatase under conditions which permit dephosphorylation of the substrate by a known phosphatase; c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
32. A kit for assaying for serine or threonine kinase activity which comprises a substrate which can be phosphorylated by a serine or threonine kinase and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate.
33 A kit for assaying for phosphatase activity which comprises a substrate having a phosphorylated serine or threonine residue and a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate.
34 A method for identifying a modulator of kinase activity, which method is a screening assay which comprises. a) providing a kinase and a substrate which can be phosphorylated by the kinase ; s b) contacting the kinase with its substrate m the presence and absence of a candidate modulator of the activity of the kinase under conditions which permit phosphorylation of the substrate by the kinase,• c) contacting the substrates treated according to step (b) 0 with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate treated according to step (b) m the presence of the s candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
35. A method for identifying a modulator of phosphatase activity which method is a screening assay which comprises : 0 a) providing a substrate having a phosphorylated residue, b) contacting the substrate with a phosphatase in the presence and absence of a candidate modulator of the activity of the phosphatase under conditions which permit dephosphorylation of the substrate by the phosphatase; 5 c) contacting the substrates treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) comparing binding of the reporter to the substrate 0 treated according to step (b) in the presence of the candidate modulator with binding of the reporter to the substrate treated according to step (b) in the absence of the candidate modulator.
36. A method for assaying for kinase activity which comprises : a) providing a substrate for a kinase,- b) contacting the substrate with a candidate kinase under conditions which permit phosphorylation of the substrate by a known kinase; c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
37. A method for assaying for phosphatase activity which comprises : a) providing a substrate having a phosphorylated residue; b) contacting the substrate with a candidate phosphatase under conditions which permit dephosphorylation of the substrate by a known phosphatase; c) contacting the substrate treated according to step (b) with a reporter, excluding a natural antibody, which binds phosphorylated substrate with higher affinity than unphosphorylated substrate; and d) monitoring binding of the reporter to the substrate treated according to step (b) .
PCT/GB2000/003736 1999-10-01 2000-09-29 Kinase and phosphatase assays WO2001025477A2 (en)

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