WO1998011251A1 - Detection de sites de reconnaissance de substrats pour les proteine kinases et les proteine phosphatases - Google Patents

Detection de sites de reconnaissance de substrats pour les proteine kinases et les proteine phosphatases Download PDF

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Publication number
WO1998011251A1
WO1998011251A1 PCT/GB1997/002466 GB9702466W WO9811251A1 WO 1998011251 A1 WO1998011251 A1 WO 1998011251A1 GB 9702466 W GB9702466 W GB 9702466W WO 9811251 A1 WO9811251 A1 WO 9811251A1
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WIPO (PCT)
Prior art keywords
ligand
peptide
enzyme
bound
library
Prior art date
Application number
PCT/GB1997/002466
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English (en)
Inventor
Shankar Balasubramanian
Christopher Abell
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Cambridge University Technical Services Ltd.
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Publication date
Application filed by Cambridge University Technical Services Ltd. filed Critical Cambridge University Technical Services Ltd.
Priority to AU41314/97A priority Critical patent/AU4131497A/en
Publication of WO1998011251A1 publication Critical patent/WO1998011251A1/fr

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Classifications

    • 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
    • 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
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • 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
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/976—Trypsin; Chymotrypsin

Definitions

  • This invention relates to a method for the detection and identification of substrate recognition sites for protein kinases and protein phosphatases.
  • Phosphatases and kinases play important metabolic roles. The ability to modulate their activity is a major focus of interest for the chemical industry. The ability to identify the substrate specificity of phosphatases and kinases is of importance inter alia for the design of inhibitors of these enzymes.
  • Protein kinases phosphorylate either tyrosine, serine or threonine residues of a protein or short peptide, whereas protein phosphatases dephosphorylate phosphotyrosine, phosphoserine or phosphothreonine residues of a protein or short peptide.
  • Methods used so far to screen substrates for phosphatases include analysing dephosphorylation by detecting a UV change, use of radioactive phosphate, colouri etric assay and ELISA.
  • Methods used so far to screen substrates for protein kinases include the use of radioactive phosphate.
  • radioactive phosphate For example, the screening of a bead-bound peptide library, by incubation with a protein kinase and radioactive ATP, has been disclosed. This results in the transfer of radioactive phosphate to those peptides which are substrates for the kinase. The radioactive beads are then isolated and the peptides sequenced.
  • a novel assay system has now been developed that can be utilised to screen a resin-bound or solution-phase phosphotyrosine peptide library for substrate turnover by a selected protein tyrosine phosphatase (PTP) or kinase.
  • PTP protein tyrosine phosphatase
  • the present invention is capable of identifying individual hits from a catalysis-based screen. It has the ability to identify individual sequences rather than simply a positional preference for particular residues. Furthermore, it has an advantage in not requiring radioactive reagents.
  • CT proteolytic enzyme ⁇ -chymotrypsin
  • the phosphopeptide substrate is synthesised on a solid phase by standard methods.
  • a non- cleavable (alkylamine) linker may typically be used.
  • a library of phosphotyrosyl peptides is screened with a phosphatase. Those phosphotyrosyl peptides which are substrates for the phosphatase will be dephosphorylated, converting the phosphotyrosine to tyrosine. Those phosphopeptides which are not substrates will not be dephosphorylated.
  • This library which now includes both phosphopeptides and dephosphorylated peptides, may then be treated with a second enzyme, chymotrypsin.
  • ⁇ -Chymotrypsin is a proteolytic enzyme that preferentially cleaves peptides adjacent to aromatic residues e.g. tyrosine groups.
  • the ⁇ -chymotrypsin cleaves adjacent to phosphotyrosine groups at a very much reduced rate with respect to non-phosphorylated tyrosine. The discrimination is so high that for the purposes of the screen it can be considered not to cleave.
  • ⁇ -chymotrypsin leaves the phosphopeptides unchanged, but cleaves those peptides which, as phosphopeptides, were substrates for the phosphatase.
  • the cleavage of the peptide can be detected in several ways, e.g.
  • This methodology can be used in connection with a FRET assay system in several ways. For example, a fluorescence donor is placed at one side of the phosphorylated site and an acceptor is placed at the other side. If the peptide is dephosphorylated on tyrosine, it becomes a substrate for ⁇ - chymotrypsin. Treatment with chymotrypsin then results in a fluorescence signal as the peptide is cleaved and the donor and acceptor separate.
  • This methodology is applicable whether the target peptide is attached to a solid support (e.g. a resin bead) or is in solution as either an individual compound or part of a mixture.
  • Preferred solid supports are Kieselguhr and PEGA (bis(2-acrylamidoprop-l-yl) polyethylene glycol cross- linked dimethyl acrylamide and acryloyl sarcosine methyl ester) resins which give excellent substrate turnover efficiencies for CT, PTP and proteins as large as 140 kDa.
  • the cleaved peptide is selected and its sequence determined. This can be achieved in several ways, e.g.
  • Sequencing by conventional peptide sequencing methods e.g. Edman degradation or mass spectrometry ; see US Patent Specification No. 5,470,753 of a sequencing strand, e.g. a sequence which has been made in parallel to the original sequence but in which the phosphotyrosine residue is replaced by a dummy residue e.g. glycine. This renders the sequencing strand non-cleavable by ⁇ -chymotrypsin. The cleaved target strand is not sequenced because the first cycle of sequencing is blocked by the fluorescent tag. This method is most appropriate when dealing with phosphopeptide libraries on solid support or single peptides in solution.
  • this system may be extended for screening kinases by running them in the reverse direction (i.e. as phosphatases) .
  • This can be achieved by treating the phosphotyrosine peptide library with a protein tyrosine kinase and ADP or water, under conditions where the kinase reaction operates in the reverse direction, to dephosphorylate the phosphopeptide and also form ATP or phosphate.
  • any cleaved dephosphorylated peptides would be detected as described above for the protein tyrosine phosphatase assay.
  • the use of a double enzyme system can be extended to other kinds of assay, e.g. assays for liposylation, ADP-ribosylation or glycosylation.
  • ⁇ - chymotrypsin is used as the proteolytic enzyme.
  • the natural amino-acids Phe, Tyr and Trp must be excluded from the peptides, since they may promote CT-mediated cleavage and thus potentially give false positives.
  • Other hydrophobic side-chains such as Leu and lie could pose a similar problem.
  • the kinetics of CT cleavage will be significantly slower than at Tyr, so optimisation of the screen should minimise false positives.
  • Example illustrates the invention, and shows that ⁇ -chymotrypsin cleaves preferentially on non- phosphorylated peptides.
  • the Example is carried out using the substrate specificity of leukocyte antigen receptor (LAR) phosphatase.
  • LAR leukocyte antigen receptor
  • the undecapeptide corresponding to the autophosphorylation site of the epidermal growth factor receptor (EGFR 988 . 998 , SEQ ID NO.l) has been shown to be a good substrate for Yersinia and mammalian PTPases, and was used as a prototype sequence for the combinatorial library.
  • EGFR 98 ⁇ . 99 ⁇ contains 3 acidic residues on the N-terminal side of the phosphorylated tyrosine (Tyr) . It is noted that several PTPs exhibit a general requirement for >4 residues on the N-terminal side of the phosphotyrosine, of which at least one should bear an acidic side-chain.
  • the phosphopeptide library was synthesised on Kieselguhr resin using a "split and mix approach", inserting one of 8 amino-acids (D, E, G, V, S, M, Q, P) in each of the positions designated X X 2 , X 3 , to generate a 512 member library. All reactions were carried out at room temperature (20°C) unless otherwise stated.
  • the Kieselguhr resin 400 mg was swollen in DMF for 30 minutes, washed with two volumes of DMF and coupled to the first amino-acid (5-fold equivalent (5 eq) ) using PyBOP (5 eq) , HOBt (5 eq) and DIPEA (10 eq) in DMF (2 ml) . The reaction was gently agitated from time to time. After 90 minutes the resin was washed (3 x DMF, 1 x DCM, 1 x DMF) . A Kaiser test was taken before the last wash. If the test was positive, then the coupling step was repeated. Otherwise the resin was treated with 20% piperidine in DMF (2 x 5 minutes) , washed as before and another Kaiser test taken. If the test proved positive, then the next residue in the sequence was coupled using the same conditions as before.
  • variable amino-acids was chosen to include acidic, hydrophobic, hydrophilic, large and small side-chains, in order to explore the requirement for carboxyl side-chains in these positions for LAR PTP.
  • the resin was lightly dried (final wash was with DCM) and poured onto a sheet of aluminium foil. It was distributed evenly between 8 reaction vessels (by weight) and the appropriate amino-acid coupling solution added to each reaction vessel as before. At the end of each coupling following a negative Kaiser result, the resins were combined for Fmoc deprotection and redistribution.
  • the terminal Fmoc was left on the sequences.
  • the final library was air-dried and then treated with a cocktail of TFA/H 2 0/TIS (95:2.5:2.5) for 3 hours, washed thoroughly, dried and stored desiccated at 4°C. Dephosphorylation
  • the library (40 mg) was swollen in buffer (30 mM Hepes, 150 mM NaCl, 6.25 mM DTT, pH 7.4, 500 ⁇ l) at 37°C for 30 minutes.
  • LAR solution 200 units, of which 1 unit will hydrolyse 1 nmole of pNPP per minute in Hepes buffer (pH 7.4) at 37°C, in 500 ⁇ l Hepes buffer
  • the reaction was then washed (water, methanol, DCM, DMF).
  • the library was allowed to equilibrate in buffer (20 mM Tris/HCl, 160 mM NaCl, pH 8.0) for 30 minutes, drained and treated with the CT solution (100 units, based on suppliers value of 52 units per mg of crystallised protein in 1 ml Tris buffer) for 45 minutes with occasional agitation. After 30 minutes, the library was washed (water, methanol, DCM, DMF). Fluorescence labelling
  • the library was allowed to equilibrate in DMF, treated with the coupling solution 5 (&6) -carboxyfluorescein (2.5 mg) , PyBOP (3.5 mg) , HOBt (1 mg) and DIPEA (2.3 ⁇ l) freshly dissolved in DMF (500 ⁇ l) , and allowed to react in the dark for 3 hours, before washing (DMF, DCM, DMF) . Fmoc removal
  • the DMF-equilibrated library was treated with a solution of 20% piperidine/DMF (2 x 5 minutes) , washed (DMF, DCM) and air-dried.
  • the library was poured out onto a Petri dish and swollen in Tris buffers. The intensities of various beads were observed by eye. There was generally a background of pale yellow beads. Deep orange beads were deemed as "hits" and removed for peptide sequencing.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO •
  • ANTI-SENSE NO
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • FRAGMENT TYPE N-terminal

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Analyse qui permet d'identifier un substrat pour une enzyme qui enlève un ligand d'un peptide auquel ledit ligand est fixé. Ce procédé peut être utilisé pour cribler une bibliothèque de peptides phosphotyrosine liés à une résine ou en phase de solution en vue d'y identifier le rendement de substrat par une protéine tyrosine phosphatase ou kinase sélectionnée.
PCT/GB1997/002466 1996-09-13 1997-09-10 Detection de sites de reconnaissance de substrats pour les proteine kinases et les proteine phosphatases WO1998011251A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU41314/97A AU4131497A (en) 1996-09-13 1997-09-10 Detection of substrate recognition of protein kinases and phosphatases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9619168.9A GB9619168D0 (en) 1996-09-13 1996-09-13 Assay
GB9619168.9 1996-09-13

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WO1998011251A1 true WO1998011251A1 (fr) 1998-03-19

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061789A1 (fr) * 1999-04-09 2000-10-19 Trustees Of Tufts College Procedes et reactifs servant a determiner la specificite de substrats enzymatiques et utilisations correspondantes
WO2000066766A1 (fr) * 1999-05-05 2000-11-09 Aurora Biosciences Corporation Sondes et analyses optiques
US7195884B2 (en) 2002-07-19 2007-03-27 Promega Corp. Methods and kits for transferases
JPWO2005071056A1 (ja) * 2004-01-23 2007-11-01 国立大学法人九州工業大学 バイオチップ及びそれを用いた試料溶液の機能性検査方法
US20090249513A1 (en) * 2005-03-07 2009-10-01 National University Corporation Nagoya University Method for Expression and Accumulation of Peptide in Plant
US7727752B2 (en) 2003-07-29 2010-06-01 Life Technologies Corporation Kinase and phosphatase assays

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993003377A1 (fr) * 1991-07-31 1993-02-18 Ziltener Hermann J Procede de detection de restes de phosphotyrosine
US5532167A (en) * 1994-01-07 1996-07-02 Beth Israel Hospital Substrate specificity of protein kinases
WO1996040276A1 (fr) * 1995-06-07 1996-12-19 Sugen, Inc. Epreuves de selection de composes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993003377A1 (fr) * 1991-07-31 1993-02-18 Ziltener Hermann J Procede de detection de restes de phosphotyrosine
US5532167A (en) * 1994-01-07 1996-07-02 Beth Israel Hospital Substrate specificity of protein kinases
WO1996040276A1 (fr) * 1995-06-07 1996-12-19 Sugen, Inc. Epreuves de selection de composes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MUNDAY ET AL: "Identification by amino acid sequence of 3 major regulatory phosphorylation sites of rat acetyl co-enzyme A carboxylase", EUR.J.BIOCHEM., vol. 175, no. 2, 1988, pages 331 - 338, XP002049597 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061789A1 (fr) * 1999-04-09 2000-10-19 Trustees Of Tufts College Procedes et reactifs servant a determiner la specificite de substrats enzymatiques et utilisations correspondantes
WO2000066766A1 (fr) * 1999-05-05 2000-11-09 Aurora Biosciences Corporation Sondes et analyses optiques
US6410255B1 (en) 1999-05-05 2002-06-25 Aurora Biosciences Corporation Optical probes and assays
JP2003503013A (ja) * 1999-05-05 2003-01-28 オーロラ バイオサイエンシズ コーポレーション 光学的プローブおよびアッセイ
AU785136B2 (en) * 1999-05-05 2006-10-05 Invitrogen Corporation Optical probes and assays
US7195884B2 (en) 2002-07-19 2007-03-27 Promega Corp. Methods and kits for transferases
AU2003253962B2 (en) * 2002-07-19 2007-08-09 Promega Corporation Methods and kits for transferases
US7314729B2 (en) 2002-07-19 2008-01-01 Promega Corp. Methods and kits for transferases
US7727752B2 (en) 2003-07-29 2010-06-01 Life Technologies Corporation Kinase and phosphatase assays
US8487078B2 (en) 2003-07-29 2013-07-16 Life Technologies Corporation Kinase and phosphatase assays
JPWO2005071056A1 (ja) * 2004-01-23 2007-11-01 国立大学法人九州工業大学 バイオチップ及びそれを用いた試料溶液の機能性検査方法
US20090249513A1 (en) * 2005-03-07 2009-10-01 National University Corporation Nagoya University Method for Expression and Accumulation of Peptide in Plant

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Publication number Publication date
AU4131497A (en) 1998-04-02
GB9619168D0 (en) 1996-10-23

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