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WO1999057305A1 - Procede detection de substances activatrices et inhibitrices de la proteine kinase b - Google Patents

Procede detection de substances activatrices et inhibitrices de la proteine kinase b Download PDF

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Publication number
WO1999057305A1
WO1999057305A1 PCT/SE1999/000609 SE9900609W WO9957305A1 WO 1999057305 A1 WO1999057305 A1 WO 1999057305A1 SE 9900609 W SE9900609 W SE 9900609W WO 9957305 A1 WO9957305 A1 WO 9957305A1
Authority
WO
WIPO (PCT)
Prior art keywords
xaa
amino acid
preferably chosen
sequence
pkb
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/SE1999/000609
Other languages
English (en)
Inventor
Thomas Olin
Stephen James
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Health AB
Original Assignee
Pharmacia and Upjohn AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmacia and Upjohn AB filed Critical Pharmacia and Upjohn AB
Priority to EP99948555A priority Critical patent/EP1090140A1/fr
Priority to CA002327540A priority patent/CA2327540A1/fr
Priority to AU42982/99A priority patent/AU754508B2/en
Priority to JP2000547256A priority patent/JP2002514389A/ja
Publication of WO1999057305A1 publication Critical patent/WO1999057305A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • 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
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention relates to method for screening for substances which are activators or inhibitors of Protein kinase B (PKB) and can be used as a kinase substrate for PKB by the use of peptides comprising a specific sequence which do not include a large hydrophobic residue at the C-terminal end.
  • PKB Protein kinase B
  • peptides comprising a specific sequence which do not include a large hydrophobic residue at the C-terminal end.
  • These peptides can be used in assays measuring the activity of PKB, in screening for substances which are activators or inhibitors of gene transcriptional regulation of forkhead proteins through the catalytic activities of PKB and for discrimination between the effects of compounds which mediate insulin action through transcription from those which modulate activity of enzymes involved in metabolism by phosphorylation.
  • the substrate peptide sequence comprises the sequence 1 ArgXaaArgXaaXaaSerXaa or sequence 2, ArgXaaArgXaaXaaThrXaa, in which Xaa in position 2 is any amino acid, preferably chosen from Pro and Gly, Xaa in positions 4 and 5 are any amino acid, preferably chosen from Thr and Ser and Xaa in position 7 is any amino acid, preferably chosen from Asn, Gin, Thr, Ser and with the proviso that the sequence does not include a large hydrophobic residue directly C-terminal to the phophorylation site.
  • insulin receptor substrate A key downstream protein in insulin signalling is phosphoinositide 3-kinase (PI3K) which catalyses the production of the second messenger phosphatidylinositol 3,4,5-trisphosphate.
  • PKB appears to be a key intermediary in the regulation of glucose utilisation and control of protein synthesis by insulin (Cross et al. 1995); (Cohen et al. 1997); (Peak et al. 1998); (Gingras et al. 1998)).
  • GSK3 glycogen synthase kinase 3
  • PKB has been shown to phosphorylate and activate phosphofructo kinase-2 (Deprezet al.
  • a third likely substrate for PKB is the type 3B cyclic AMP phosphodiesterase (Wijkander et al. 1998), which in insulin-responsive tissues is activated by phosphorylation, leading to the inactivation of adrenergic-stimulated processes.
  • Figure la Cos-7 transfected with HA-PKB. Phosphorylation of Crosstide (black bar) compared with a peptide according to the invention (grey bar)
  • Figure lb Inactive rPKB ⁇ activated by incubation with IGF-1 stimulated muscle cell lysate. Phosphorylation of Crosstide (black bar) compared with a peptide according to the invention (grey bar)
  • PKB Protein kinase B
  • the peptides can be used for discrimination between the effects of compounds which mediate insulin action through transcriptional regulation from those compounds which modulate activity of enzymes involved in metabolism by phosphorylation. Such a discrimination has not been possible earlier. 4
  • the invention relates to the use of a peptide sequence comprising the sequences
  • ArgXaaArgXaaXaaSerXaa sequence 1, or ArgXaaArgXaaXaaThrXaa, sequence 2.
  • the sequence can be included as a part of any peptide or protein provided that the sequence is accessible to the targeting enzyme PKB.
  • the peptide is preferably SerThrPheArgProArgThrSerSerAsnAla, sequence 14.
  • amino acids Asp, Glu, Lys and Arg are charged and could possibly have an influence on phosphorylation.
  • large or strongly hydrophobic residue is for example meant Phe, Leu, He, Trp and
  • Gene transcriptional regulation involves activation or repression of the enzymes and other components involved in metabolism, for example the repression of PEPCK
  • FKHRL1, AFX and AF6q21 (Accession number AF032885, AF032886, X939996 and
  • peptides as defined in the claims can be used in the search for new substrates for PKB as templates for sequence searches and/or for primers in techniques of molecular biology such as PCR.
  • Example 1 The isoform of PKB was immunoprecipitated from lysates of Cos-7 cells (approx. lmg of protein) transfected with bovine PKB ⁇ which contained an N-terminal haemagglutinin (HA) tag. After washing, phosphorylation of the peptides RPRTSSF (black bar in Figure la) and STFRPRTSSNA (grey bar in Figure la) by PKB was measured in parallel assays by incubation in the presence of 33 P-labelled ATP. Comparison of results from the two peptides showed that within a 30 minute assay, equal amounts of peptide were converted to the phosphorylated form at a rate of 0.5pmol per minute under the assay conditions employed. 5
  • the example suggest that the peptide of the invention is useful for determining PKB activity of other isoforms than the ⁇ -form.
  • peptide substrates for use in PKB phosphorylation assays were refined by investigating the ability of recombinant activated PKB to phosphorylate various different peptides ( Figures 2a and b).
  • peptides (lOO ⁇ M) were incubated with 0.2 ⁇ g activated recombinant PKB (purchased from UBI) in the presence of 1 O ⁇ M 33 P-ATP and the extent of phosphorylation was compared with that of the peptide sequence RPRTSSF, previously used as a substrate for PKB (Alessi et al, 1996).
  • Figure 2a shows that addition of up to three neutral or small hydrophobic amino acids to either end of the sequence PKB is thought to phosphorylate had no marked effect on the extent of phosphorylation of each respective peptide.
  • peptides KKRNRTLTK (a peptide often used to measure the activity of a kinase related to PKB, p70 S6 kinase) and APRPRVETSQ (derived from pyruvate dehydrogenase kinase 1) were phosphorylated to less than one quarter the extent of GRPRTSSF by PKB.
  • APRPRVETSQ derived from pyruvate dehydrogenase kinase 1
  • the compounds found from the claimed screening are anticipated also to be used against long term complications resulting from insulin resistance, such as vascular dysfunction, loss of neuronal cells and ⁇ -cells in pancreas. These compounds are not possible to find by using the modulators as described in WO 97/22360. 7

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

Abstract

L'invention concerne un procédé permettant de détecter des substances activatrices et inhibitrices de la protéine kinase B (PKB), qui peuvent être utilisées comme substrat de kinase pour la PKB, par l'intermédiaire de peptides comprenant une séquence spécifique, qui contient pas une grande quantité de résidu hydrophobe, au niveau de l'extrémité terminale C. Ces peptides peuvent être utilisés dans des dosages permettant de mesurer l'activité de la PKB, dans la recherche de substances activatrices ou des inhibitrices de régulation transcriptionnelle génique de protéines en tête de fourche, à travers les activités catalytiques de la PKB, et pour une discrimination entre les effets des composés qui régulent l'action de l'insuline, à travers une transcription à partir des composés qui modulent l'activité des enzymes impliquées dans un métabolisme par phosphorylation. La séquence de peptide de substrat comprend la séquence 1 ArgXaaArgXaaXaaSerXaa ou une séquence 2 dans laquelle Xaa en position 2 représente un acide aminé quelconque, choisi de préférence à partir de Pro et Gly; Xaa en position 4 et 5 représe un acide aminé quelconque, choisi de préférence à partir de Thr et Ser; et Xaa en position 7 représente un acide aminé quelconque, choisi de préférence à partir de Asn, Gln, Thr, Ser à condition que la séquence ne contienne pas une grande quantité de résidu hydrophobe
PCT/SE1999/000609 1998-04-30 1999-04-16 Procede detection de substances activatrices et inhibitrices de la proteine kinase b Ceased WO1999057305A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP99948555A EP1090140A1 (fr) 1998-04-30 1999-04-16 Procede detection de substances activatrices et inhibitrices de la proteine kinase b
CA002327540A CA2327540A1 (fr) 1998-04-30 1999-04-16 Procede detection de substances activatrices et inhibitrices de la proteine kinase b
AU42982/99A AU754508B2 (en) 1998-04-30 1999-04-16 Method for screening for substances which are activators or inhibitors of Protein kinase B
JP2000547256A JP2002514389A (ja) 1998-04-30 1999-04-16 プロテインキナーゼｂの賦活物質または阻害物質となる物質のスクリーニング方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9801530-8 1998-04-30
SE9801530A SE9801530D0 (sv) 1998-04-30 1998-04-30 Method for screening

Publications (1)

Publication Number Publication Date
WO1999057305A1 true WO1999057305A1 (fr) 1999-11-11

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1999/000609 Ceased WO1999057305A1 (fr) 1998-04-30 1999-04-16 Procede detection de substances activatrices et inhibitrices de la proteine kinase b

Country Status (6)

Country Link
EP (1) EP1090140A1 (fr)
JP (1) JP2002514389A (fr)
AU (1) AU754508B2 (fr)
CA (1) CA2327540A1 (fr)
SE (1) SE9801530D0 (fr)
WO (1) WO1999057305A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006023879A1 (fr) * 2004-08-20 2006-03-02 Board Of Regents, The University Of Texas System Selection d'agents presentant une activite contre les accidents ischemiques myocardiques
US7491702B2 (en) 2001-04-18 2009-02-17 The Open University Polypeptides related to amyloid precursor protein, pharmaceutical compositions thereof, and methods of treatment using the same
US7622446B2 (en) 2001-04-18 2009-11-24 The Open University Polypeptides, derivatives and uses thereof
US8158586B2 (en) 2005-04-11 2012-04-17 Pharmagap Inc. Inhibitors of protein kinases and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018823A2 (fr) * 1994-01-07 1995-07-13 Beth Israel Hospital Specificite de substrat des proteines kinases
WO1997018303A1 (fr) * 1995-11-16 1997-05-22 Novartis Ag Proteine kinase rac utile en tant qu'agent therapeutique ou dans des diagnostics
WO1997022360A2 (fr) * 1995-12-20 1997-06-26 Medical Research Council Regulation de synthese de proteines et procede de criblage pour agents pharmaceutiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018823A2 (fr) * 1994-01-07 1995-07-13 Beth Israel Hospital Specificite de substrat des proteines kinases
WO1997018303A1 (fr) * 1995-11-16 1997-05-22 Novartis Ag Proteine kinase rac utile en tant qu'agent therapeutique ou dans des diagnostics
WO1997022360A2 (fr) * 1995-12-20 1997-06-26 Medical Research Council Regulation de synthese de proteines et procede de criblage pour agents pharmaceutiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DARIO R. ALESSI ET AL.: "Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase", FEBS LETTERS, vol. 399, 1996, pages 333 - 338 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7491702B2 (en) 2001-04-18 2009-02-17 The Open University Polypeptides related to amyloid precursor protein, pharmaceutical compositions thereof, and methods of treatment using the same
US7622446B2 (en) 2001-04-18 2009-11-24 The Open University Polypeptides, derivatives and uses thereof
WO2006023879A1 (fr) * 2004-08-20 2006-03-02 Board Of Regents, The University Of Texas System Selection d'agents presentant une activite contre les accidents ischemiques myocardiques
US7531318B2 (en) 2004-08-20 2009-05-12 Board Of Regents, The University Of Texas System Screening of agents for activity against ischemic myocardial insults
US8158586B2 (en) 2005-04-11 2012-04-17 Pharmagap Inc. Inhibitors of protein kinases and uses thereof

Also Published As

Publication number Publication date
AU4298299A (en) 1999-11-23
AU754508B2 (en) 2002-11-21
SE9801530D0 (sv) 1998-04-30
JP2002514389A (ja) 2002-05-21
EP1090140A1 (fr) 2001-04-11
CA2327540A1 (fr) 1999-11-11

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