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CA2327540A1 - Method for screening for substances which are activators or inhibitors of protein kinase b - Google Patents

Method for screening for substances which are activators or inhibitors of protein kinase b Download PDF

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Publication number
CA2327540A1
CA2327540A1 CA002327540A CA2327540A CA2327540A1 CA 2327540 A1 CA2327540 A1 CA 2327540A1 CA 002327540 A CA002327540 A CA 002327540A CA 2327540 A CA2327540 A CA 2327540A CA 2327540 A1 CA2327540 A1 CA 2327540A1
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Prior art keywords
xaa
amino acid
preferably chosen
sequence
pkb
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CA002327540A
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French (fr)
Inventor
Thomas Olin
Stephen James
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Swedish Orphan Biovitrum AB
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    • 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

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

Abstract

The present invention relates to methods 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. 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, 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, Gln, Thr, Ser and with the proviso that the sequence does not include a large hydrophobic residue directly C-terminal to the phosphorylation site.

Description

Method for screening for substances which are activators or inhibitors of Protein Icinase B
Summary 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. 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 ~ 5 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 ArgXaa.ArgXaaXaaSerXaa or sequence 2, ArgXaaArgXaaXaaThrXaa, in which Xaa in position 2 is any amino 2o 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, Gln, Thr, Ser and with the proviso that the sequence does not include a large hydrophobic residue directly C-terminal to the phophorylation site.
25 Background The mechanistic basis by which insulin regulates the disposition of glucose by animals has been elucidated in close detail in recent years. A cascade of interacting proteins has been described which, when functioning in the normal situation, serve to transduce the signals emanating from insulin, causing cells of different origin to take up glucose 3o from the bloodstream and store it (White 1997). In the current understanding, activation of the insulin receptor by insulin causes the phosphorylation and activation of insulin receptor substrate (IRS) proteins. These serve to act as docking proteins for a variety of downstream proteins leading to their activation. 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.
This is a lipid and which is central to the activation of PKB ((Franke et al. 1995);
(James et al.
1996); (Franke et al. 1997); (Klippel et al. 1997); (Alessi et al. 1997);
(Stokoe et al.
1997)). It is bound by the pleckstrin homology (PH) domains of PKB and of an upstream kinase called 3-phosphoinositide-dependent kinase 1 (PDK 1 ) which is involved in the activation of PKB.
t o 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)). Thus, it has been demonstrated to phosphorylate and inactivate glycogen synthase kinase 3 (GSK3; (Cross et al. 1995)), permitting the synthesis of glycogen from glucose. Furthermore, in cardiac myocytes, PKB has been 1 s shown to phosphorylate and activate phosphofructo kinase-2 (Deprezet al.
1997) whose product, fructose 2,6-bisphosphate, acts as an allosteric activator of glycolysis.
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.
Although the range of substrates phosphorylated by PKB is diverse, all of those described share a common short primary sequence which serves as the target region for PKB. The first identified consensus sequence of amino acids was in GSK-3, described as GlyArgProAxgThrSerSerPheAIaGluGly (GRPRTSSFAEG ) {Cross et al. 1995). See also WO 97/22360.
Comparison with other substrates allows a consensus sequence to be derived from this which is likely to contain the essential features for phosphorylation by PKB.
This consensus is: RXRXXS/TF (Alessi et al. 1996) where F is phenylalanine but could be replaced by another bulky hydrophobic residue. Data in the literature (not only Alessi 3o et al. 1996 but also Walker et al. 1998) strongly suggest that the amino acid sequence for phophorylation by PKB must include a large hydrophobic residue directly C-terminal to the phophorylation site.
Such a residue has thus been described as crucial for phosphorylation by PKB.
Studies in C. elegans have recently suggested that an important effect of insulin may be to suppress the transcriptional activity in the family of transcription factors (Ogg et al. 1997). By inactivating a specific transcription factor in C. elegans the authors have shown that C. elegans can recover metabolism in the absence of the insulin receptor.
Figures Figure la: Cos-7 transfected with HA-PKB. Phosphorylation of Crosstide (black bar) compared with a peptide according to the invention (grey bar) o Figure lb: Inactive rPKBa 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) Figure 2a. Phosphorylation of peptides compared with that of the peptide sequence RPRTSSF.
~ 5 Figure 2b. Phosphorylation of peptides with different modifications compared with that of the peptide sequence GRPRTSSF.
The invention 2o We have now found peptides which do not include a large hydrophobic residue directly C-terminal to the phophorylation site.
These peptides have unexpectedly been shown to be as good substrates for PKB
as the earlier known peptides with the large hydrophobic residue directly C-terminal to the phophorylation site.
25 These novel peptides can be used in screening for substances which are activators, inhibitors or binders of Protein kinase B (PKB).
The invention is defined in the attached claims.
The peptides can be used for discrimination between the effects of compounds which mediate insulin action through transcriptional regulation from those compounds which 3o modulate activity of enzymes involved in metabolism by phosphorylation.
Such a discrimination has not been possible earlier.

WO 99!57305 PCT/SE99/00609 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.
(STFRPRTSSNA).
The sequences used in the screening method are defined in the attached claims.
The amino acids Asp, Glu, Lys and Arg are charged and could possibly have an influence on phosphorylation.
t o By large or strongly hydrophobic residue is for example meant Phe, Leu, Ile, Trp and Cys.
By PKB any isoform thereof is included.
The uses of the defined sequences are defined in the attached claims.
Gene transcriptional regulation involves activation or repression of the enzymes and other components involved in metabolism, for example the repression of PEPCK
(phosphoenol pyruvate carboxykinase) in liver.
Members of the forkhead transcription factor family can be exemplified by FKHR, FKHRL1, AFX and AF6q21 (Accession number AF032885, AF032886, X939996 and AJ001589, respectively).
2o Furthermore the 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 I
The a isoform of PKB was immunoprecipitated from lysates of Cos-7 cells (approx.
lmg of protein) transfected with bovine PKBa which contained an N-terminal haemagglutinin (HA) tag. After washing, phosphorylation of the peptides RPRTSSF
(black bar in Figure 1 a) and STFRPRTSSNA (grey bar in Figure 1 a) by PKB was measured in parallel assays by incubation in the presence of 33P-labelled ATP.
3o 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.
As immunoprecipitation of PKB can co-precipitate other forms of the enzyme and COS7 cells contain wildtype PKB, the current assay will comprise other isoforms than PKBa.
Hence, the example suggest that the peptide of the invention is useful for determining PKB activity of other isoforms than the a-form.
Example 2 Recombinant, virtually inactive GST-PKBa, pre-coupled to glutathione beads was incubated and activated with increasing concentrations of lysate from IGF-1-1 o stimulated H9C2 cardiac myocytes (approx. 0, 20, 100 and 200pg/ml) in the presence of unlabelled ATP. The beads were subsequently washed thoroughly and used to phosphorylate each of the peptides RPRTSSF (black bar in Figure lb) and STFRPRTSSNA (grey bar in Figure lb) in the presence of 3'P-labelled ATP. Data showed that each peptide was equivalently phosphorylated by PKB and that each t s peptide was phosphorylated to the same extent as the degree of activation of the enzyme increased.
This experiment proves that the peptide of the invention is effective for assaying activity of the PKBa isoform.
20 Example 3 The general features of 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). Thus, peptides (100p.M) were incubated with 0.2p.g activated recombinant PKB (purchased from UBI) in the presence of 25 l Op.M "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.
These data 3o further reinforce our assertion that peptides derived from the fork head transcription factor family of proteins which lack a bulky hydrophobic amino acid C-terminal to the phosphorylated residue are good substrates for PKB.
Figure 2b shows the ability of PKB to phosphorylate peptides with different modifications. Addition of a bulky unit such as a biotin moiety to the N-terminal end of the peptide GRPRTSSF reduced phosphorylation by PKB by 75%. In addition, 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 phosphoryiated to less than one quarter the extent of GRPRTSSF by PKB. These data indicate that addition of charged amino acids to either end of the consensus peptide sequence phosphorylated by PKB markedly 1 o reduces the ability of PKB to phosphorylate them. In particular, each peptide contains a charged residue immediately adjacent to the amino acid which becomes phosphorylated, indicating that PKB may favour no charge at these positions, directly N- and C-terminal to the targetted residue.
Discussion.
Our data show that the reported requirement for a large hydrophobic amino acid residue directly C-terminal to the targetted residue for PKB-mediated phosphorylation is not necessary for an efficient phosphorylation, and that most amino acids are 2o suitable in the same position (as seen in FKHR and other members of the transcription factor family) without compromising the ability of PKB to phosphorylate.
The data also show that our claimed novel peptides can be used as an efficient component in the screening for PKB modulators.
The claimed peptides can be used for finding compounds useful for treating patients 2s having a deficiency of the amount of essential components in metabolism such as transducers of the insulin signalling pathway and enzymes involved in metabolism.
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 (3-cells in pancreas. These compounds are not 3o possible to find by using the modulators as described in WO 97/22360.

WO 99/S730S PCTlSE99/00609 7 _ References Alessi, D.et al. (1996).FEBS Letters 399: 333-338.
Alessi, D. Ret al. ( 1997). Current Biology 7: 261-269.
Cohen, Pet al. (1997). FEBS Letters 410: 3-10.
Cross, D., et al. ( 1995). Nature 378: 785-789.
Deprez, Det ai. (1997).Journal of Biological Chemistry 272: 17269-17275.
Franke, T. F, et al. (1997).Science 275: 665-668.
Franke, T. F et al. (1995). Cell 81: 727-736.
Gingras, A.-Cet al. (;1998). Genes and Development 12: 502-513.
1 o James, S. R., et al. ( 1996). Biochemical Journal 315: 709-713.
Klippel, A.,et al. (1997). Molecular and Cellular Biology 17: 338-344.
Ogg, S. et al. ( 1997). Nature 389: 994-999.
Peak, M., J. et al. ( 1998). Diabetologia 41: 16-25.
Stokoe, D. et al. (1997). Science 277: 567-570.
Walker, K. S. et al. (1998). Biochemical Journal 331: 299-308.
White, M. F. (1997). Diabetologia 40: S2-S17.
Wijkander, J. et al. ( 1998). Endocrinology 139: 219-227.

1 _ SEQUENCE LISTING
<110> Pharmacia & Upjohn AB
<120> Method for screening for substances which are activators or inhibitors of Protein kinase B
<130> List <140>
<141>
<150> SE9801530-8 <151> 1998-04-30 <160> 14 <170> PatentIn Ver. 2.0 <210> 1 <211> 7 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 1 Arg Xaa Arg Xaa Xaa Ser Xaa <210> 2 <211> 7 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 2 Arg Xaa Arg Xaa Xaa Thr Xaa <210> 3 <211> 7 <212> PRT
<213> Homo sapiens <400> 3 Arg Pro Arg Thr Ser Ser Asn <210> 4 <211> 8 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 4 Xaa Arg Xaa Arg Xaa Xaa Ser Xaa <210> 5 <211> 8 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 5 Xaa Arg Xaa Arg Xaa Xaa Thr Xaa <210> 6 <211> 8 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 6 Arg Xaa Arg Xaa Xaa Ser Xaa Xaa <210> 7 <211> 8 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 7 Arg Xaa Arg Xaa Xaa Thr Xaa Xaa <210> 8 <211> 9 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 8 Xaa Arg Xaa Arg Xaa Xaa Thr Xaa Xaa <210> 9 <211> 9 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 9 Xaa Arg Xaa Arg Xaa Xaa Thr Xaa Xaa <210> 10 <211> 9 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 10 Phe Arg Xaa Arg Xaa Xaa Ser Xaa Xaa <210> 11 <211> 9 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 11 Phe Arg Xaa Arg Xaa Xaa Thr Xaa Xaa <210> 12 <211> I1 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 12 Ser Thr Phe Arg Xaa Arg Xaa Xaa Ser Xaa Xaa <210> 13 <211> 11 <212> PRT
<213> Unknown <220>
<223> Description of Unknown Organism: Not known <400> 13 Ser Thr Phe Arg Xaa Arg Xaa Xaa Thr Xaa Xaa <210> 14 <211> 11 <212> PRT
<213> Homo Sapiens <400> 14 Ser Thr Phe Arg Pro Arg Thr Ser Ser Asn Ala

Claims (17)

8
1. Method for screening for substances which are activators or inhibitors of Protein kinase B (PKB) by the use of a substrate peptide comprising the sequences 1 or ArgXaaArgXaaXaaSerXaa (1) or ArgXaaArgXaaXaaThrXaa (2) 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, Gln, Thr, Ser and with the proviso that the sequence does not include a large hydrophobic residue directly C-terminal to the phophorylation site.
2. Method for screening according to claim 1 with the proviso that Xaa in positions 4 and 5 is not chosen from Asp, Glu, Lys or Arg.
3. Method for screening according to claim 1 or 2 in which the peptide consists of the primary sequence 3 ArgProArgThrSerSerAsn (3)
4. Method for screening according to claim 1 in which the peptide comprises the sequence 4 or 5 XaaArgXaaArgXaaXaaSerXaa (4) or XaaArgXaaArgXaaXaaThrXaa (5) in which Xaa in position 1 is a large hydrophobic amino acid, preferably Phe.
Xaa in position 3 is any amino acid, preferably chosen from Pro and Gly, Xaa in positions 5 and 6 are any amino acid, preferably chosen from Thr and Ser and Xaa in position 8 is any amino acid, preferably chosen from Asn, Gln, Thr, Ser and with the proviso that the sequence does not include a large hydrophobic residue directly C-terminal to the phophorylation site.
5. Method for screening according to claim 4 with the proviso that Xaa in positions 5 and 6 is not chosen from Asp, Glu, Lys or Arg.
6. Method for screening according to any of claims 1 to 3 in which the peptide comprises the sequence 6 or 7 ArgXaaArgXaaXaaSerXaaXaa (6) or ArgXaaArgXaaXaaThrXaaXaa (7) in which Xaa in position 8 is a small hydrophobic amino acid, preferably chosen among Ala and Gly.
7. Method for screening according to any of claims 4 to 6 in which the peptide comprises the sequence 8 or 9 XaaArgXaaArgXaaXaaSerXaaXaa (8) or XaaArgXaaArgXaaXaaThrXaaXaa (9) in which Xaa in position 9 is a small hydrophobic amino acid, preferably chosen among Ala and Gly.
8. Method for screening according to claim 7 in which the peptide comprises the sequence 10 or 11 PheArgXaaArgXaaXaaSerXaaXaa (10) or PheArgXaaArgXaaXaaThrXaaXaa (11)
9. Method for screening according to claim 7 in which the peptide comprises the sequence 12 SerThrPheArgXaaArgXaaXaaSerXaaXaa (12) in which Xaa in position 5 is any amino acid, preferably chosen from Pro and Gly, Xaa in positions 7 and 8 are any amino acid, preferably chosen from Thr and Ser and Xaa in position 10 is any amino acid, preferably chosen from Asn, Gln, Thr, Ser Xaa in position 11 is a small hydrophobic amino acid, preferably chosen among Ala and Gly.
10. Method for screening according to claim 8 in which the peptide comprises the primary sequence 13 SerThrPheArgXaaArgXaaXaaThrXaaXaa (13) in which Xaa in position 5 is any amino acid, preferably chosen from Pro and Gly, Xaa in positions 7 and 8 are any amino acid, preferably chosen from Thr and Ser and Xaa in position 10 is any amino acid, preferably chosen from Asn, Gln, Thr, Ser Xaa in position 11 is a small hydrophobic amino acid, preferably chosen among Ala and Gly.
11. Method for screening according to claim 9 in which the peptide consists of the primary sequence 14 SerThrPheArgProArgThrSerSerAsnAla (14)
12. Method for screening for substances which are activators, inhibitors and binders of gene transcriptional regulation by forkhead proteins through the catalytic activities of PKB characterised by the use of any of the peptides defined in any of claims 1-11.
13. Method for screening for substances which have the capacity to modulate PKB
activity characterised by the use of any of the peptides defined in any of claims 1-11.
14. Use of a peptide as defined in any of claims 1-11 in assays measuring the activity of PKB.
15. Use of a peptide as defined in any of claims 1-11 in screening for substances which are activators or inhibitors of gene transcriptional regulation by forkhead proteins through the catalytic activities of PKB.
16. Use of the peptide as defined in any of claims 1-11 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.
17. Use according to claim 14 for discrimination between the effects of compounds which mediate insulin action through transcription via forkhead transcription factor family from those which modulate activity of enzymes involved in metabolism by phosphorylation.
CA002327540A 1998-04-30 1999-04-16 Method for screening for substances which are activators or inhibitors of protein kinase b Abandoned CA2327540A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9801530A SE9801530D0 (en) 1998-04-30 1998-04-30 Method of screening
SE9801530-8 1998-04-30
PCT/SE1999/000609 WO1999057305A1 (en) 1998-04-30 1999-04-16 Method for screening for substances which are activators or inhibitors of protein kinase b

Publications (1)

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EP (1) EP1090140A1 (en)
JP (1) JP2002514389A (en)
AU (1) AU754508B2 (en)
CA (1) CA2327540A1 (en)
SE (1) SE9801530D0 (en)
WO (1) WO1999057305A1 (en)

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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
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

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US5532167A (en) * 1994-01-07 1996-07-02 Beth Israel Hospital Substrate specificity of protein kinases
EP0862622B1 (en) * 1995-11-16 2004-08-18 Novartis AG Method of screening for RAC protein kinase interacting compounds
CA2241080A1 (en) * 1995-12-20 1997-06-26 Medical Research Council Control of protein synthesis, and screening method for agents

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WO1999057305A1 (en) 1999-11-11
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AU4298299A (en) 1999-11-23
AU754508B2 (en) 2002-11-21
SE9801530D0 (en) 1998-04-30

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