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WO1994008600A1 - Inhibition de molecules transductrices de signaux - Google Patents

Inhibition de molecules transductrices de signaux Download PDF

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Publication number
WO1994008600A1
WO1994008600A1 PCT/US1993/009626 US9309626W WO9408600A1 WO 1994008600 A1 WO1994008600 A1 WO 1994008600A1 US 9309626 W US9309626 W US 9309626W WO 9408600 A1 WO9408600 A1 WO 9408600A1
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seq
met
peptide
glu
analog
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Steven Shoelson
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Joslin Diabetes Center Inc
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Joslin Diabetes Center Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the inhibition of signal transduction molecules.
  • 5 Protein tyrosine kinases including, e.g., growth factor receptors, proto-oncogene and oncogene products, and the insulin receptor, catalyze tyrosine phosphorylations within their own sequences as well as on other cellular proteins. These phosphorylations are thought to be essential for transmission of ligand binding signals into cells and for intermolecular interactions within cells. The regulation of o tyrosine phosphorylation levels appears to be important for both cellular growth and metabolic control.
  • PTPases Protein-tyrosine phosphatases
  • PTKs Protein-tyrosine phosphatases
  • Families of PTPases are now being identified e.g., by cloning and expression of DNA sequences homologous to the catalytic domains of placental 5 PTPase IB and LCA/CD45 from leukocytes, which are among the best characterized PTPases.
  • the invention features peptides capable of inhibiting an interaction, preferably a site specific interaction, of an SH2-domain-containing 0 protein, e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the insulin receptor or the PDGF receptor, or a protein which is active in the regulation of cell proliferation, e.g., an SH2-domain-containing 0 protein, e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the insulin receptor or the PDGF receptor, or a protein which is active in the regulation of cell proliferation, e.g., an SH2-domain-containing 0 protein, e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein,
  • R 1 1 is Met, Val, He, or Glu
  • R ⁇ 7 is any amino acid, but is preferably Pro, Met, Asp, Thr, Asn, Glu, or Tyr
  • Tyr-Met-R 2 -Met SEQ ID NO: 1
  • Tyr-R 1 -R 2 -R 3 (SEQ ID NO: 2) (wherein R 1 is Glu, Asp, Thr, Tyr His, or Gin; R ⁇ is Glu, Asn, Tyr, or Asp; and R J is He, Met, Leu, or Val) e.g., Tyr- Glu-Glu-Ile, (SEQ ID NO: 3) e.g., a protein capable of altering the state of phosphorylation of a tyrosine residue, e.g., a tyrosine kinase.
  • Peptides of the inventio 0 include the sequence R -R -R -R 4 (SEQ ID NO: 4) (wherein Ri is tyrosine, phosphotyrosine, or more preferably, an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g. phosphonomethylphenylalanine (Pmp), or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, mono- or
  • R J is any amino acid, but is preferably He,
  • the peptide includes the sequence R -R -R -Met (SEQ ID NO: 5)
  • Ri is phosphotyrosine or, more preferably, an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g. phosphonomethylphenylalanin (Pmp), or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, FPmp or F2Pmp;
  • R is Met, Val, He, or Glu; and
  • Rx is any amino acid, but is preferably Pro, Met, Asp, Thr, Asn, Glu, or Tyr
  • Pmp-Met-R 3 -Met SEQ ID NO: 6
  • FPmp-Met-R -Met SEQ ID NO: 6
  • F2Pmp-Met-R 3 -Met SEQ ID NO: 6
  • the peptide includes the sequence R 1 -R 2 -R 3 -R 4 (SEQ ID NO: 4) (wherein Ri is phosphotyrosine or, more preferably, an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g.
  • phosphonomethylphenylalanine or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, FPmp or F2Pmp;
  • R is Glu, Asp, Thr, Tyr, His, or Gin;
  • R 3 is Glu, Asn, Tyr, or Asp; and
  • R is He, Met, Leu, or Val) e.g., Pmp-Glu-Glu-Ile (SEQ ID NO: 7), FPmp-Glu-Glu-Ile (SEQ ID NO: 7) or F2Pmp-Glu-Glu-Ile (SEQ ID NO: 7).
  • the peptide is between 4 and 30 amino acids in length; the peptide is between 4 and 15 amino acids in length; the peptide is at least 40%, preferably at least 80%, and more preferably at least 95% homologous, with a segment of a naturally occurring protein which interacts with an SH2 containing protein, e.g., tyrosine phosphatase substrate.
  • the invention features a method of inhibiting an interaction, preferably a site specific interaction, between a first molecule which includes an SH2 domain, e.g., a signal transduction protein, e.g., a cytoplasmic or a transmembrane signal transduction protein, a receptor protein, e.g., the insulin receptor or the PDGF receptor, or a protein which is active in the regulation of cell proliferation, e.g., an oncogene product, and a second molecule, e.g., a protein containing the sequence Tyr-R 1 -R 7 -Met (SEQ ID NO: 1) (wherein R 1 1 is Met, Val,
  • R is any amino acid, but is preferably Pro, Met, Asp, Thr, Asn, Glu, or Tyr) e.g., Tyr-Met-R 2 -Met (SEQ ID NO: 8), or the sequence Tyr-R 1 -R 2 -R 3 (SEQ ID NO: 8).
  • the method includes contacting the first molecule with an inhibitor molecule which includes a peptide of the invention.
  • the first molecule is a molecule which transmits a signal, e.g., an extracellular signal, across a membrane an the second molecule is an enzyme which can alter the phosphorylation state of tyrosine, e.g., a tyrosine kinase; the first molecule is an oncogene protein and the second molecule is an enzyme which can alter the phosphorylation state of tyrosine, e.g., a tyrosine kinase; the first molecule is the insulin receptor and the second molecule is an enzyme which can alter the phosphorylation state of tyrosine, e.g., a 5 tyrosine kinase; the inhibitor molecule inhibits the first molecule from binding to the second molecule; the inhibitor molecule inhibits the phosphorylation of the first molecule; the inhibitor inhibits the binding of the first molecule to a third molecule; the inhibitor results in an alteration of a catalytic
  • the invention features a method of treating a mammal, e.g., a human, having a condition characterized by unwanted cell proliferation including administering to the mammal an amount of a peptide of the invention sufficient to prevent or inhibit the unwanted cell proliferation.
  • the peptide prevents the association of an SH2 domain containing oncogene with a second molecule, e.g., a protein containing the sequence Tyr-R 1 -R - Met (SEQ ID NO: 1) (wherein R 1 is Met, Val, He, or Glu; and R 2 is any amino acid, but is preferably Pro, Met, Asp, Thr, Asn, Glu, or Tyr) e.g., Tyr-Met-R 2 -Met (SEQ ID NO: 1) (wherein R 1 is Met, Val, He, or Glu; and R 2 is any amino acid, but is preferably Pro, Met, Asp, Thr, Asn, Glu, or Tyr) e.g., Tyr-Met-R 2 -Met (SEQ ID NO: 1) (wherein R 1 is Met, Val, He, or Glu; and R 2 is any amino acid, but is preferably Pro, Met, Asp, Thr, Asn, Glu, or Tyr) e.g., Tyr-Met-R 2
  • Tyr-Glu-Glu-Ile (SEQ ID NO: 3), e.g., a protein capable of altering the state of phosphorylation of a tyrosine residue, e.g., a tyrosine kinase.
  • the invention features a method of synthesizing a peptide, e.g., a peptide of the invention, containing a phosphonomethylphenylalanine residue 5 including: providing a phosphonomethylphenylalanine preferably, with a protected phosphonate side chain; and incorporating the phosphonomethylphenylalanine into th peptide.
  • the method further includes removing the protection group from the phosphonate side chain after the o phosphonomethylphenylalanine has been incorporated into the peptide; and the protected phosphonate side chain is protected by a t-butyl group present as a t-butyl ester.
  • the invention features a peptide analog of a protein tyrosine phosphatase substrate, the substrate including a tyrosine residue.
  • the analog 5 includes an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g., a phosphonomethylphenylalanine residue, or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, FPmp or F2Pmp; in place of a tyrosine residue and is capable of inhibiting dephosphorylation of the substrate by a tyrosine phosphatase.
  • the analog includes one of the peptides of the invention described above, e.g.,the sequence Pmp-Met-Rl-Met (SEQ ID NO: 6),
  • FPmp-Met-Ri-Met SEQ ID NO: 6
  • F2Pmp-Met-R 1 -Met SEQ ID NO: 6
  • Pmp is phosphonomethylphenylalanine Ri is any amino acid; the analog is between 4 and 30, and more preferably between 4 and 15, amino acids in length.
  • the analog is at least 40%, preferably at least 80%, and more preferably at least 95% homologous with a naturally occurring tyrosine phosphatase substrate.
  • the analog has the sequence RDIPmpETDYYRK (SEQ ID NO: 9), RDIYETDPmpYRK (SEQ ID NO: 10), RDIYETDYPmpRK (SEQ ID NO: 11), TEPEPmpQPGE (SEQ ID NO: 12), KDESIDPmpVPMLDMKGD (SEQ ID NO: 13), RENEPmpMPMAPQIH (SEQ ID NO: 14), TDDGPmpMPMSPGV (SEQ ID NO: 15), GNGDPmpMPMSPKS (SEQ ID NO: 16), RDI[L-Pmp]ETDYYRK (SEQ ID NO: 17), RDI[D-Pmp]ETDYYRK (SEQ ID NO: 18), RDIPmpETDPmpPmpRK (SEQ ID NO: 19), RDH ⁇ PmpETDYYRK (SEQ ID NO: 9), RDIYETDF2PmpYRK (SEQ ID NO: 10), RDIYETDYF P
  • KDESIDF2PmpVPMLDMKGD (SEQ ID NO: 13), RENEF2PmpMPMAPQIH (SEQ ID NO: 14), TDDGF2PmpMPMSPGV (SEQ ID NO: 15), GNGDF 2 PmpMPMSPKS (SEQ ID NO: 16), RDI[L-F 2 Pmp]ETDYYRK (SEQ ID NO: 17), RDI[D- F Pmp]ETDYYRK (SEQ ID NO: 18), RDIF2PmpETDF2PmpF 2 PmpRK (SEQ ID NO: 19), RDIFPmpETDYYRK (SEQ ID NO: 9), RDIYETDFPmpYRK (SEQ ID NO:
  • RDIYETDYFPmpRK SEQ ID NO: 11
  • TEPEFPmpQPGE SEQ ID NO: I2
  • KDESIDFPmpVPMLDMKGD SEQ ID NO: 13
  • RENEFPmpMPMAPQIH SEQ I NO: 14
  • TDDGFPmpMPMSPGV SEQ ID NO: 15
  • GNGDFPmpMPMSPKS SEQ ID NO: 16
  • RDI[L-FPmp]ETDYYRK SEQ ID NO: 17
  • RDI[D-FPmp]ETDYYRK SEQ ID NO: 18
  • RDIFPmpETDFPmpFPmpRK SEQ ID NO: 19
  • the invention also includes a method of inhibiting the dephosphorylation o a substrate by a protein tyrosine phosphatase.
  • the method includes contacting the protein tyrosine phosphatase with an inhibiting amount of an analog of a naturally occurring protein tyrosine phosphatase substrate, the analog including an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g., phosphonomethylphenylalanine or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, FPmp or F2Pmp.
  • a hydrolysis resistant phosphorous moiety e.g., phosphonomethylphenylalanine or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, FPmp or F2Pmp.
  • the peptide is between 4 and 30 amino acids in length; the peptide is between 4 and 15 amino acids in length; the peptide is at least 5 40%, preferably at least 80 %, and more preferably at least 95% homologous, with a segment of a naturally occurring protein tyrosine phosphatase substrate.
  • the invention also includes a method of treating a mammal e.g., a human, afflicted with a disease or disorder characterized by the dephosphorylation of a protein tyrosine phosphatase substrate.
  • the method includes administering to the o mammal an inhibiting amount of a peptide analog of a naturally occurring protein tyrosine phosphatase substrate, the analog including an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g., phosphonomethylphenylalanine or a hydrolysis resistant phosphorous moiety which i more electronegative than the phosphate group of phosphotyrosine, for example, 5 FPmp or F2Pmp.
  • a mammal e.g., a human, afflicted with a disease or disorder characterized by the dephosphorylation of a protein tyrosine phosphatase substrate.
  • the method includes administering to the o mammal an inhibiting amount
  • the invention includes methods of increasing the affinity of a phosphopeptide for its substrate, e.g., a protein containing a SH2-domain.
  • the method includes replacing the phosphate moiety of a phosphotyrosine with a moiety which is more electronegative than the phosphate moiety of phosphotyrosine such as 0 in R 1 -OP03H2 where R 1 can be CHF, CF 2 , CHC1, CC1 2 , or CC1F.
  • the invention includes peptides which have been modified to make them more resistant to proteolytic degradation and include e.g., depsipeptide derivatives of the peptides disclosed herein, e.g., peptides which have been modified by the reduction of amide bonds, the inclusion of D-amino acids, or end methylation.
  • Signal transduction protein refers to a protein involved in transferring a signal from the cell surface into the cell and includes, e.g., membrane bound receptors, e.g., cell surface receptors, ligands of such receptors, and intracellular proteins which interact with either with a receptor, or with another intracellular protein to transfer a signal.
  • SH2 domain refers to a conserved apparently noncatalytic sequence of approximately 100 amino acids found in many signal transduction proteins including Fps, Stc, Abl, GAP, PLC ⁇ , v-Crk, Nek, p85, and Vav. See Koch e al., 1991, Science 252:668. hereby incorporated by reference. The amino acid sequences of the SH2 domain of 27 proteins is given in Koch et al., 1991.
  • the SH2 5 domain mediates protein-protein interactions between the SH2 containing protein and other proteins by recognition of a specific site on a second protein. The SH2/second protein site interaction usually results in an association of the SH2 contacting protein and the second protein.
  • SH2 domain refers to any sequence with at least 70%, preferably at least 80%, and more preferably at least 90% sequence homology with a naturally occurring SH2 domain, and to any analog or fragment of a SH2 domain which exhibits at least 50% of the binding activity of a naturally occurring variant of that domain, when binding is measured as the ability to bind a YM M (SEQ ID NO: 20) containing peptide.
  • An interaction between an SH2 domain containing protein and a second molecule e.g., a protein refers to any of: binding characterized by noncovalent or covalent interactions; an interaction which includes the alteration of the phosphorylation state of either the SH2 domain containing or another molecule, e.g., the second molecule; or to an interaction which includes an alteration of a catalytic ability of the SH2 domain containing proteins, or another molecule, e.g., the second molecule.
  • Analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety refers to an amino acid with a side chain having a moiety of the formula
  • R is a moiety which renders the phosphate group more resistant to ⁇ ⁇ ⁇ enzymatic hydrolysis than would be the case if the R was O.
  • R and R are preferably H.
  • a preferred example of R is CR 4 R5, wherein R 4 is H, or a small electronegative atom, e.g., F or Cl, and R ⁇ is H or a small electronegative atom, e.g., F or Cl.
  • Particularly preferred embodiments of the invention include hydrolysis resistant phosphorous moieties that are more electronegative than the phosphate moiety of phosphotyrosine, for example, where R is -CHF, -CF2, -CCIF, -CHCl, or
  • Increasing the electronegativity of the phosphate moiety in an analog of phosphotyrosine increases the binding affinity of peptides containing the analog to a substrate such as protein containing an SH2 domain. In some cases, the affinity can exceed that of peptides containing phosphotyrosine.
  • mT polyoma virus middle T antigen
  • mT/pp60 c-src polyoma virus middle T antigen/pp60 c_src complex
  • Ptdlns 3 -kinase phosphatidylinositol 3-(hydroxy) kinase
  • Ptdlns phosphatidylinositol
  • PtdIns-3-P phosphatidylinositol 3-phosphate
  • PtdIns-4-P phosphatidylinositol 4-phosphate
  • PtdIns-4,5-P2 phosphatidylinositol 4,5-bisphosphate
  • PtdIns-3,4-P2 phosphatidylinositol 3,4-bisphosphate
  • PtdlnsP phosphatidylnsP
  • SH2 domain containing proteins are involved in cellular signaling, e.g., in the signal transduction mediated by insulin and the insulin receptor and by several classes of oncogenes.
  • the invention provides for inhibitors of these cellular signal transduction systems by inhibiting an interaction between the SH2 domain of the signal transduction protein and a YM ⁇ M (SEQ ID NO: 20) motif present on another protein.
  • the invention provides for interference with the transduction of growth signals and thereby allows for control of unwanted cellular proliferation.
  • the invention also provides peptide inhibitors of PTPases.
  • the peptide inhibitors, or analogs which may or may not have homology with naturally occurring protein tyrosine phosphatase substrates, include an analog of phosphotyrosine having a hydrolysis resistant phosphorous moiety, e.g., phosphonomethylphenylalanine or a hydrolysis resistant phosphorous moiety which is more electronegative than the phosphate group of phosphotyrosine, for example, FPmp or F2Pmp.
  • the phosphotyrosine analog includes a phenyl group substituted with an -O-PO3H2 analog, e.g., -R-PO3H2, where R can be any group which confers greater resistance to hydrolysis than does -0-, e.g, CR 4 R ⁇ wherein R 4 can be H, F or Cl and R ⁇ can be H, F or Cl.
  • Preferred phosphotyrosine analogs are phosphonomethylphenylalanine and mono- or difluorophosphonomethylphenylalanine.
  • Peptide inhibitors of the invention can have homology with PTPase substrates. The sequence of these inhibitors can be based on the amino acid sequences of kinase autophosphorylation and endogenous substrate phosphorylation sites.
  • the invention provides for the incorporation of Pmp into peptides using derivatives of Fmoc-Pmp in which the P-OH groups are protected as t-butyl esters. This procedure minimizes the yield of impurities arising from side reactions with free P-OH groups.
  • Pmp-peptides are useful, e.g., as nonhydrolyzable inhibitors and affinity ligands of proteins having phosphotyrosine as part of a recognition element.
  • Phosphonomethylphenylalanyl peptides and mono- and difluorophosphonomethylphenylalanyl peptides constitute new classes of compounds 5 that potently inhibit PTPase activity.
  • Pmp-peptides appear to act as direct substrate mimics, as binding affinity closely matches that of the corresponding phosphopeptide and inhibition is competitive.
  • F2Pmp peptides also appear to act as direct substrate mimics. However, binding affinity of the F2Pmp-peptides can match or exceed that o the corresponding phosphopeptides.
  • Inhibitors of the invention allow the inhibition o 0 cellular PTPases and can be used in controlling metabolic processes, e.g., abnormal processes associated with diabetes, and as therapeutic modalities for selected malignancies. The inhibitors are also useful to study the enzymatic mechanisms of
  • PTPase activity and to investigate the metabolic and biochemical roles of PTPases.
  • FIG. 1 is a diagram of Fmoc-Pmp (tBu)2-OH (Fig. 1 A) and two Pmp- containing peptides (Fig IB (SEQ ID NO: 44) and 1C (SEQ ID NO: 21)).
  • Fig. 2 is a graph of the results of a binding study.
  • Phosphotyrosine ( ⁇ ) replaced in the peptide GXVPML (SEQ ID NO: 45) by Pmp (D), monofluorophosphonomethylphenylalanine (FPmp) (0), F2Pmp, (star), 5 hydroxyfluorophosphonomethylphenylalanine (HPmp) (O), and tyrosine (Tyr) ( ⁇ ).
  • T ED50 values were 0.17 ⁇ 0.02 ⁇ M for pTyr, l.O ⁇ O.l ⁇ M for Pmp, 0.50 ⁇ 0.03 ⁇ M for FP 0.17 ⁇ 0.02 ⁇ M for F2Pmp, 3.3 ⁇ 0.5 ⁇ M for HPmp, and >l,000 ⁇ M for Tyr.
  • Fig. 3 is a depiction of: panel A, the binding affinity of the peptide DXVPML (SEQ ID NO:46) for the SH2 domain of PI 3-kinase where X was pTyr (•) 0 or F2Pm ⁇ (O), the ED50 values were 0.15 ⁇ 0.03 ⁇ M pTyr and 0.17 ⁇ 0.02 ⁇ M for
  • F2Pmp panel B, the binding affinity of the peptide QXEEIP (SEQ ID NO:47) for the SH2 domain of Src where X was pTyr (•) or F2Pmp (O), the ED50 values were 5.7 ⁇ 0.7 ⁇ M for pTyr and 1.0 ⁇ 0.2 ⁇ M for F2Pmp; panel C, the binding affinity of the peptide NXVNIE (SEQ ID NO: 48) for the SH2 region of Grb2 where X was pTyr (•), L-F2Pmp(0), or D-F2Pmp(D), the ED50 values were 0.9 ⁇ 0.1 ⁇ M for pTyr,
  • Fig. 4 is a diagram of the peptide GXVPML (SEQ ID NO: 45) where X in the diagram can be CH2, CHF, CF2, CHOH.
  • the phosphate group (-XPO3H2) can b OH.
  • Phosphonomethylphenylalanine is a non-natural analogue of phosphotyrosine in which the >C-0-P03H2 moiety of is replaced by >C-CH2- PO3H2. Unlike the phenyl phosphate ester of phosphotyrosine, the carbon- phosphorus bond of Pmp is stable to chemical and enzyme-catalyzed hydrolysis. Incorporation of Pmp into specific peptide sequences yields phosphopeptide analogs which are stable to the actions of cellular PTPases. Pmp has been prepared previously, Marseigne et al., 1988, J. Org. Chem.
  • Racemic N ⁇ -Fmoc-(0,0-di-t-butyl)phosphono-p-methylphenylalanine (Fmoc-Pmp(tBu)2-OH) (see Fig. 1 A) was prepared in three steps from ⁇ -(0,0-di-t- butylphosphono)-/?-tolualdehyde, Burke et al., 1991, J. Med. Chem., 24:1577, in 48% overall yield, Burke et al., Synthesis.
  • the new derivative was used to synthesize inhibitory peptides, e.g., the 11 -amino acid peptide corresponding to a mono- phosphorylated sequence of the insulin receptor (residues 1155-1165), Ebina et al., 1985, Cell, 40:747; White et al., 1988, J. Biol. Chem., 262:2969, (see Fig. IB) (SEQ ID NO: 44) and the 13 -amino acid peptide corresponding to a putative phosphorylation site of the hamster polyoma virus transforming protein, middle T, Delmas et al., 1985, EMBO J., 4:1279, (see Fig. 1C) (SEQ ID NO: 21).
  • inhibitory peptides e.g., the 11 -amino acid peptide corresponding to a mono- phosphorylated sequence of the insulin receptor (residues 1155-1165), Ebina e
  • Peptides from both HPLC fractions gave identical results by plasma desorption mass 0 spectrometry, (results from plasma desorption mass spectrometry (ABI Biolon): D- and L-isomers, the insulin receptor peptide (SEQ ID NO: 9) molecular ions at m/z 1601.7 and 1601.8; D- and L-isomers.
  • IRS-1 is an endogenous substrate of the insulin receptor that associates with PI 3-kinase following insulin stimulation of intact cells, Sun et al., 1991, Nature 252:73. Peptides corresponding to suspected IRS-1 phosphorylation sites were 5 prepared. YMZM (SEQ ID NO: 20) sequences were found to be phosphorylated much more efficiently than other sequences. Methionine residues at the Y + 1 and Y + 3 positions of the YMZM (SEQ ID NO: 6) motif are necessary for directing insulin kinase action efficiently. Phosphopeptides corresponding to YM ⁇ M (SEQ ID NO: 20) sequences and autophosphorylation sites of the insulin receptor and src were l o prepared.
  • Phosphorylated YMZM (SEQ ID NO: 20) peptides had the unique ability to inhibit interaction of IRS- 1 with PI 3-kinase activity. Unphosphorylated YMZM (SEQ ID NO: Q ⁇ ) sequences and other phosphopeptides had no effect.
  • o Peptides were phosphorylated by insulin and ATP-activated receptors in a time and temperature dependent fashion to display saturable kinetics and linear Lineweaver-Burk plots.
  • V ma ⁇ peptide phosphorylation
  • K m peptide concentrations required for half-maximal saturation
  • Vmax or kcat ancl overa11 catalytic efficiency (k ca t K m ) all of the YMZM peptide sequences are excellent substrates of the insulin receptor kinase. YZZM-peptides were phosphorylated less efficiently.
  • positions within peptide Y987 were substituted and subjected to similar kinase assays. Met Y + 1 substitutions (He, Thr) reduced catalytic efficiency (k ca t/K m ) nearly 5-fold, while norleucine, whose side 5 chain mimics that of methionine regarding both hydrophobicity and flexibility, had no observable effect.
  • Fmoc-pTyr(OMe)2 or Fmoc-pTyr(OBzl)2 can be used to prepare phosphorylated forms of YMZM peptides.
  • Phosphopeptides having a mouse middle antigen sequence (pY-midT, EEEpYMEMEDLY (SEQ ID NO: 32)), sequences from the kinase insert of the PDGF receptor (pY708, DGGpYfcr ⁇ MSKDE (SEQ ID NO: 33); pY719, SIDpYVPMLDMK (SEQ ID NO: 34)) and IRS-1 YMZM (SEQ ID NO: o 20) sequences pY608 (SEQ ID NO: 25), pY628 (SEQ ID NO: 26), pY658 (SEQ ID NO: 27), pY727 (SEQ ID NO: 13), pY939 (SEQ ID NO: 29) and pY987 (SEQ ID NO: 30) (Table 1) have all been prepared, purified and characterized by amino acid analysis and FAB-MS.
  • IRS-1 Inhibiting interactions between IRS-1 and PI 3-kinase IRS-1 produced in a 5 baculovirus expression system was adsorbed to anti-peptide (IRS-1) antibodies and phosphorylated by solubilized insulin receptors (which were then washed away).
  • Ptdlns 3-kinase was reconstituted with recombinant, baculovirus-expressed, immunopurified mT/pp60 c_src in vitro.
  • the Ptdlns 3-kinase associated with protein-tyrosine kinase-active mT/pp60 c_src but failed to associate with an inactive mutant.
  • ATP was not required during the precipitation indicating that phosphorylation of Ptdlns 3-kinase itself was not needed for tight complex formation.
  • Murine mT EEEEYMPMEDLYL 315 (SEQ ID NO: 36)
  • Rat IRS- 1 TDDGYMPMSPGVA 608 (SEQ ID NO: 37)
  • Rat IRS- 1 APVSYADMRTGIA 1010 (SEQ ID NO: 39)
  • the non-phosphorylated peptide had no effect on the association.
  • the phosphotyrosine os this sequence is analogous to tyr-315 of murine mT, the major sit of tyrosine phosphorylation in vivo.
  • Mutations in the region of tyr-315 also compromise the transforming ability o polyoma virus without affecting the tyrosine kinase activity of the mT/pp60 c_src complex, indicating that this region is critical for in vivo targeting of the protein- tyrosine kinase, Cantley et al., 1991, Cell, 64:281. (These experiments are discussed in detail below).
  • receptor-type tyrosine kinases that bind Ptdlns 3- kinase have short stretches of sequences with similarity to the tyr-315 region of mT, Cantley et al., 1991, supra.
  • IRS-1 insulin receptor tyrosine 1
  • a major substrate of the insulin receptor tyrosine contains several repeats of sequences similar to the tyr-315 region of polyoma mT, Sun et al., 1991, Nature, 352:73. (Table 2).
  • This protein is not a tyrosine kinase but associates with Ptdlns 3-kinase in insulin-stimulated (but not unstimulated) cells, Sun et al., 1991, supra. IRS-1 has two regions of sequence that are quite similar to the hamster mt phosphopeptide used for competition studies reported herein (Table 2).
  • the results presented here provide the first evidence that the 110 kDa subunit of Ptdlns 3-kinase associates with the mT/pp60 c-src complex.
  • the 110 kDa subunit was not phosphorylated as well as the 85 kDa subunit in the reconstituted complex and was not previously detected in 3? P ⁇ 43 ⁇ labeled proteins associated with mT immunoprecipitates from polyoma-infected cells, Kaplan et al., 1987, Cell, 50: 1021.
  • the 85 kDa protein has twO ⁇ 80 amino acid stretches of homology 5 to the amino-terminal non-catalytic domain of pp60 c_src (SH-2 domain), Ostu et al., 1991, Cell, 65:91; Escobedo et al., 1991, Cell, Little:75; Skolnik et al., 1991, Cell, Little:83.
  • SH-2 domains have been shown to bind to tyrosine-phosphorylated regions of target proteins to form tight complexes both in vivo and in vitro (Anderson et al. (Pawson) 1990, reviewed in Cantley et al., 1991, supra, and (Pawson's Science review 1991)).
  • Tyrosine-phosphorylated mT specifically blots the 85 kDa (but not the 110 kDa) subunit of Ptdlns 3-kinase using a Western blot procedure, Carpenter et al., 1990, supra; Cohen et al., 1990, supra.
  • the mT/pp60 c_src complex has been generated in insect Sf9 cells by co-infection with baculovirus constructs that express the mT and c-src genes, Piwnica- Worms et al., 1990, supra. This complex has been shown to have protein-tyrosine kinase activity. Both mT and pp60 c_src in 5 the insect cell and the purified complex has tyrosine kinase activity toward exogenous substrates. A kinase-defective complex, mT/pp60 2y5c_src , has also been previously characterized.
  • the mutant protein associates with mT, but does not have protein tyrosine kinase activity, Piwnica- Worms et al., 1990, supra.
  • the mT/pp60 c_src and mT/pp60 complexes were purified from baculovirus infected Sf9 cells using anti-middle T ( ⁇ -mT) antibodies conjugated to protein-A Sepharose beads.
  • the purified complexes lacked Ptdlns 3-kinase activity, although a small amount of PtdlnsP kinase activity was detected.
  • a monoclonal antibody (EC 10) directed against pp60 c_src also immunoprecipitated the mT/pp60 c"src complex and associated lipid kinase activity.
  • the gel electrophoresis analysis of phosphatidylinositol kinase reactions after association of purified rat liver Ptdlns 3-kinase with baculovirus-expressed, immunopurified mT/pp60 c-src and mT/pp60 2 " 5c"src was performed as follows. The mT complexes were immunopurified as described herein and incubated with or 5 without purified Ptdlns 3 -kinase at 4°C for 2 hours in standard lysis buffer.
  • Phosphatidylinositol kinase assays were done with a mixture of PtdS, Ptdlns, Ptdlns- 4-P, and PtdIns-4,5-P2 as lipid substrates (1 :1 :1:2). Immunopurified, baculovirus- expressed mT/pp60 c"src and mT/pp60 295c_src were used. Purified Ptdlns 3-kinase was added or was omitted as herein and incubated with or without purified Ptdlns 3- 0 kinase at 4°C for 2 hours in standard lysis buffer.
  • Phosphatidylinositol kinase assays were done with a mixture of PtdS, Ptdlns, PtdIns-4-P, and PtdIns-4,5-P2 as lipid substrates (1 :1 :1 :2). Immunopurified, baculovirus-expressed mT/pp60 c_src and mT/pp60 c were used. Purified Ptdlns 3-kinase was added or was omitted.
  • Sf9 cell pellets Preparation of Sf9 cell pellets, lysates, and immunoprecipitates was as 5 follows. Sf9 cells were scrape-harvested from the cell culture plates in phosphate buffered saline (PBS) and collected by centrifugation. The cell pellet was washed once with cold PBS and aliquoted to 1.5 ml micro-centrifuge tubes and again separated by centrifugation. The supernatant was aspirated from the cells and the pellets were quick-frozen in dry ice/ethanol and stored at -70° C until the lysates were 0 prepared.
  • PBS phosphate buffered saline
  • Lysates were generated by addition of 1.0 ml of standard lysis buffer (137 mM NaCl, 20mM Tris-HCl (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% NP-40, 150 ⁇ g/ml aprotinin and leupeptin) and incubated at 4° C with constant rocking for 15 minutes. Lysates were cleared by centrifugation at 12,000 x g for 5 5 minutes at 4° C. The mT/pp60 c_src complex was purified by immunoprecipitation essentially as described, Auger et al., 1990, Methods in Inositide Research, 159.
  • ⁇ -mT antibodies were added to the lysates and incubated at 4° C for 2 hours with constant agitation.
  • the immune complexes were collected on protein A- Sepharose (CL 4B; Sigma) that had been pre-washed in 1% BSA and then stored in a o 50% suspension with PBS.
  • Immune complexes were washed 2 times with 1% NP-40 in PBS, 2 times with RIPA (20 mM HEPES (pH 7.5), 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), twice with 0.5 M LiCl in 0.1 M Tris-HCl (pH 7.4) and finally 2 times with TNE (10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA). The immuniprecipitates were used immediately for in 5 vitro phosphorylation and association assays. Purification of phosphatidylinositol 3 -kinase was as follows.
  • Ptdlns 3- kinase was purified to homogeneity from rat liver as previously described, Ca ⁇ enter et al., 1990, supra.
  • the purified enzyme had a specific activity of approximately 500 nmol/mg/min when assayed as described below with Ptdlns as the substrate.
  • the 5 enzyme was stored to 4° C in 50 mM MES (pH 6.7), 100 mM KCl, 0.5 mM DTT, 1 ⁇ g/ml of leupeptin and pepstatin A and used within two weeks of purification.
  • Mammalian whole cell lysates were prepared from confluent 10 cm cell 5 culture plates. The monolayer was washed once with 4° C PBS and lysed in 1.0 ml of standard lysis buffer. After 10 minutes of incubation on a rocker platform at 4° C, the cells were scrape harvested and lysates cleared by centrifugation at 12,000 x g for 5 minutes at 4° C. Lysates were used immediately for association experiments with the immunopurified mT/pp60 c ⁇ src complex. o Phosphatidylinositol kinase assays and protein kinase assays were performed as follows.
  • Phosphatidylinositol kinase assays were performed directly on the immune complex as described, Whitman et al., 1985, supra; Auger et al., 1989, supra. Sonicated phospholipids were added to the washed beads and the reaction was initiated with the addition of 50 ⁇ M ATP, 5-25 ⁇ Ci [ ⁇ 32 P]ATP (3000/Ci/mmole), 5 5 mM MgCl2 in 20 mM HEPES (pH 7.5). Reactions were incubated for 5 minutes at room temperature. The 50 ⁇ l reactions were stopped by the addition of 80 ⁇ l of 1 M HC1 and 1 ⁇ l of 500 mM EDTA. The lipids were extracted with 160 ⁇ l of methanol hloroform (1: 1) and the organic layer was collected for analysis.
  • Protein kinase assay were also performed directly on the immune complex 0 with or without added Ptdlns 3-kinase. The reaction was done in 20 mM HEPES (pH
  • Phosphoamino acid analysis was performed as follows. Phosphoamino acid analysis of the [ J ⁇ 7 , P]-labelled proteins from the SDS-gel was performed as described, Parker et al., 1991, Embo J., 10:1255. Briefly, radioactive gel slices were excised after autoradiography, fixed in 30% methanol for several hours and dried to roto- evaporation. The gel slices were treated with TPCK trypsin (Worthington
  • NIH/3T3 cells were grown to approximately 80-90% confluence in 10 cm plates in DME supplemented to contain 10% CS.
  • the culture medium was removed and replaced with 5 ml of DME minus methionine (Gibco) that contained 500 ⁇ Ci Tran S-label (ICN, > 1000 Ci/mmole) supplemented with one third of the amount of unlabeled methionine normally used.
  • the cells were cultured for 12-48 hours and then harvested as follows: Radioactive culture medium was aspirated and the cell monolayer was washed 2 times with ice-cold PBS.
  • the cells were lysed by the addition of 1 ml of standard lysis buffer (see above) and incubated on a rocker platform for 15-20 minutes at 4°C.
  • the lysate was harvested with the aid of a cell scraper and cleared of insoluble material by centrifugation at 12,000 x g for 5 minutes at 4°C.
  • the lysates were used immediately for association assays.
  • Sf9 cells were labelled at 38-40 hours post-infection with Tran 35 S-label as described, Piwnica- Worms et al., 1990, supra.
  • Thin layer chromatography, deacylation of phospholipids, and HPCL analysis were performed as follows. Intact phospholipids were analyzed on oxalate treated silica gel 60 plates (E.
  • NIH/3T3 and Rat-1 fibroblasts were maintained by standard cell culture techniques in DME supplemented to contain 10% calf serum (CS).
  • Spodoptera frugiperda (Sf9) cells were cultured and used as described by Piwnica- Worms et al., 1990, supra.
  • Sf9 cells were seeded into 60-mm plates (3 x 10 cells), allowed to attach, and then were coinfected with baculovirus- mT and baculovirus-pp60 c"src , or baculovirus-pp60 rc as described, Piwnica-
  • Ptdlns 3-kinase is a heterodimer of 85 kDa and 110 kDa proteins, Ca ⁇ enter et al., 1990, supra.
  • the 85 kDa subunit has been shown to be phosphorylated on tyrosine in vivo in mT transformed cells, Kaplan et al., 1987, supra; Ca ⁇ enter et al., 1990, supra.
  • protein kinase reactions were performed directly with the immunoprecipitated complex. Protein kinase reactions were performed using immunopurified mT/pp60 c-src complex in the presence or absence of purified Ptdlns 3 -kinase. The protein kinase reaction was initiated with the addition of [ ⁇ - 39 P]ATP and stopped after 10 minutes with the addition of 2x gel loading buffer. Polyacrylamide gel electrophoresis
  • the immunopurified mT/pp60 c_src will also associate with Ptdlns 3-kinase from fibroblast lysates.
  • NIH/3T3 or Rat-1 Lysates were prepared from confluent cells in standard lysis buffer as described above. Immunopurified mT/pp60 c_src complexes were added to the whole cell lysates and incubated at 4 ⁇ C.
  • the protein A Sepharose- conjugate mT/pp60 c_src complex was then washed with detergent and salt, and o assayed for associated phospholipid kinase activity.
  • the resultant samples were extracted and deacylated for analysis by HPLC anion exchange chromatography. Ptdlns, PtdIns-4-P, and PtdIns-4,5-P2 kinase activities were present.
  • the HPLC migration positions of the three products were consistent with the structure PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3.
  • the ability of the mT/pp60 c-src to associate with Ptdlns 3 -kinase from whole cell lysates is also dependent on an active protein-tyrosine kinase as demonstrated with the purified enzyme.
  • the amount of Ptdlns 3 -kinase activity that associated with the mT/pp60 c" 5 src complex was dependent on the amount of lysate in the incubation. Titration of Rat-1 lysates against a fixed amount of mT/pp60 c_src complex revealed the immune complex was in excess and lysate from approximately 2.5 x 10 cells represented 50% saturation.
  • NIH/3T3 cells were labelled with Tran 35 S-label and whole cell lysates were prepared.
  • Immunopurified protein A-conjugated mT/pp60 c"src and mT/pp60 2y5c”src were added to separate radiolabelled lysates and incubated at 4° C for 3 hours.
  • the complexes were washed with NP-40, LiCl, and TNE.
  • the associated proteins were analyzed by SDS-PAGE. Although a number of radiolabelled peptides associate with both the active and inactive protein-kinase complexes, the most striking difference was observed in the 85 KDa and the 110 kDa regions.
  • a band at 153 kDa also preferentially associated with the active mT/pp60 c_src .
  • the phosphorylated version of the peptide could also block the association of Ptdlns 3 -kinase activity with the immunopurified mT/pp60 c_src when purified Ptdlns 3 -kinase was used for the association assay. Proteins that were prevented from associating with the mT/pp60 c_src complex by the phosphorylated peptide were also determined.
  • Rat-1 cells were pre-incubated with phosphorylated peptide, unphosphorylated peptide, or no peptide, and then association with exogenously added mT/pp60 c"src was done as described in materials and methods.
  • Gel electrophoresis experiments showed that bands consistent with Ptdlns 3-kinase (a doublet around 110 kDa and an 85 kDa band) associate with the kinase active complex but not with the protein kinase deficient mT/pp60 c .
  • the unphosphorylated peptide did not affect this association. However, the phosphorylated peptide specifically blocked three proteins from associating with the kinase active mT/pp60 c_src complex. Other, higher molecular weight proteins that associated in a protein kinase dependent manner were not blocked by the phosphorylated peptide.
  • the association between the pp60 c" S rc /polyoma virus middle T antigen (mT) complex and phosphatidylinositol 3-kinase (PI 3-kinase) can be used as a prototype for phosphoprotein-SH2 domain interactions. It can be used to test whether a peptide, e.g., a non-hydrolyzable phosphonopeptide, can inhibit the association between an SH2 domain containing protein and a YMXM (SEQ ID NO: 20) containing protein.
  • a peptide e.g., a non-hydrolyzable phosphonopeptide
  • phosphonomethylphenylalanine is a non-natural analogue of phosphotyrosine in which the >C-0-P03H2 moiety is replaced by >C-CH2-P ⁇ 3H2-
  • a thirteen amino acid phosphonopeptide mT- Pmp315), a related phosphopeptide (mT-pY315) and an unmodified sequence (mT-
  • the phosphopeptide and L-phosphonopeptide bound tightly (KTJ « 10-20 nM) and specifically to isolated SH2 domains of PI 3-kinase p85, demonstrating that the mechanism of inhibited association is competitive binding to PI 3 -kinase SH2 domains.
  • the appropriate phosphonopeptide sequence inhibits the interaction between a tyrosine-phosphorylated protein and a cognate SH2 domain- containing protein, and is resistant to the actions of PTPases.
  • Proteolytically stable phosphonopeptide derivatives are useful inhibitors of protein-protein interactions when introduced into cells, and provide a basis for the rational design of a new class of chemotherapeutic agent.
  • the sequence of the peptides is H-Glu-Glu-Glu-Xxx- Met-Pro-Met-Glu-Asp-Leu-Tyr-OH, (SEQ ID NO: 43) wherein Xxx is the targeted tyrosine residue and is substituted at the para position with any of a free hydroxyl, a phosphate or the non-hydrolyzable, methylene-bridged phosphonate.
  • Both the phosphopeptide and the phosphonopeptide were found to bind to isolated SH2 domains of the P85 subunit of PI 3-kinase. However, the in the absence of PTPase inhibitors only the phosphonopeptide inhibited pp60 c" ⁇ rc /mT complex association with cytosolic PI 3 -kinase activity.
  • Peptides were cleaved from the resin and side chain protecting groups were simultaneously removed by treatment with trifluoroacetic acid, thioanisole, ethanedithiol and anisole (90:5:3:2) for 2 h at 22°C.
  • Methyl protecting groups on phosphotyrosine were removed during a second stage of deprotection with trimethylsilyl bromide (Kitas et al., 1991, Helv. Chim. Ada 24:1314-1328). All peptides were precipitated with diethyl ether (4°C) and desalted on a column of Bio- gel P2.
  • Balb/3T3 cells were treated with lysis buffer (137 mM NaCl, 20 mM Hepes, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, and 1 ⁇ g/ml each of leupeptin, pepstatin A and aprotinin, pH 7.25) in the presence or absence of 0.15 mM sodium vanadate.
  • lysis buffer 137 mM NaCl, 20 mM Hepes, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, and 1 ⁇ g/ml each of leupeptin, pepstatin A and aprotinin, pH 7.25
  • lysis buffer 137 mM NaCl, 20 mM Hepes, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, and 1 ⁇ g
  • Lysate-peptide mixtures were combined with protein A-Sepharose beads containing immunoprecipitated pp60 c" ⁇ rc /mT complexes. After washing, the beads were used to catalyze phosphatidylinositol phosphorylation as described (Whitman et al., 1985, Nature 215:239-242; Auger et al., 1989, Cell 51:167-175, 1990, Methods in Inositide Res. pp. 159-166). Sonicated phospholipids were added to the beads and phosphorylation reactions were initiated with 50 ⁇ M [ ⁇ - 39 P]ATP, 5 mM MgCl2 in 20 mM HEPES, pH 7.5.
  • Glutathione- agarose 25 ⁇ l of a 1 :4 aqueous slurry, Molecular Probes was added and the samples were incubated at 22°C for 4 h. Following centrifugation for 5 min at 12,000 x g, supernatant solutions were removed by aspiration and [ I]radioactivity associated with the pellets was determined with a ⁇ -counter.
  • Amino Acid Side Chain pK was determined as follows. Phosphonomethylphenylalanine and phosphotyrosine were suspended in water and brought into solution by addition of sodium hydroxide to yield an 0.2 M solution, pH >10.3. Aliquots (10 ⁇ l) of 6 N HCl were added with mixing and the pH was recorded. Inhibition of PI 3 -kinase association with the pp60 c" ⁇ rc /mT complex was determined as follows. Cytosolic PI 3-kinase activity associates with pp60 c" S rc /mT complexes and can be immunoprecipitated with antibodies against either pp ⁇ O 0- ⁇ 0 or mT.
  • a phosphopeptide and matched non-hydrolyzable phosphonopeptide corresponding to the sequence surrounding Tyr315 of mouse polyoma mT were prepared as described herein.
  • both peptides inhibited association between pp60 c" ⁇ rc /mT and PI 3-kinase, with ID50 values of « 100 nM and «800 nM for the phosphopeptide and racemic phosphonopeptide, respectively.
  • Precipitated protein complexes were used to catalyze phosphorylation of phosphatidylinositol as described herein.)
  • An unphosphorylated peptide having the same hamster mT sequence had no detectable activity under these conditions (Auger et al., 1992, J. Biol. Chem. 2£Z:5408-5415).
  • phosphonopeptide inhibition of association between baculovirus-expressed mT/pp ⁇ O ⁇ 10 complex and cytosolic PI 3-kinase activity in the presence and absence of sodium vanadate peptides were incubated first with cytosol from Balb/3T3 cells at 4°C for 30 min in the presence and absence of 200 ⁇ M sodium vanadate, were combined with baculovirus-expressed mT/ p ⁇ ⁇ ' complex, anti-mT antibodies and protein A-Sepharose for an additional 2 h; peptide concentrations were chosen to inhibit ⁇ one-half of associated PI 3-kinase activity (e.g., 75-100 nM mT- pY315 ; 0.75- 1.O ⁇ M mT-Pmp315).
  • Pmp is unaltered by prolonged incubation with high concentrations of expressed PTPases and is stable under much harsher hydrolytic conditions (e.g., boiling 6.0 N HCl for 18 h; Marseigne & Roques, 1988, J. Org. Chem. 52:3621).
  • ID50 values were determined as the concentration of unlabeled peptide
  • N-terminal p85 SH2/GST fusion protein [ i "T]phosphopeptide and varying concentrations of unlabeled peptides were combined in 200 ⁇ l total volume of 20 mM Tris-HCl, 250 mM NaCl, 0.1% bovine serum albumin, lOmM dithiotheotol, pH 7.4. Glutathione- agarose (25 ⁇ l of a 1:8 aqueous slurry) was added and the samples were incubated at 22°C for 4 h. Following centrifugation for 5 min at 12,000 x g, supernatant solutions were removed by aspiration and [ 9 J 5I]radioactivity associated with the pellets was determined with a ⁇ -counter.)
  • L-mT-Pmp315 binds to the N-terminal SH2 domain of p85 2- fold weaker than the corresponding phosphopeptide (which contains L- phosphotyrosine), whereas D-mT-Pmp315 binds to the N-terminal SH2 of p85 much more weakly with a relative ID50 value «30- to 40-fold higher than the phosphopeptide.
  • Phosphopeptides were synthesized by solid-phase methods known to those skilled in the art (DIPCDI/HOBt couplings in DMF) using Fmoc-pTyr with an unprotected phosphate side chain. In many cases the desired products were obtained as single major products, particularly when pTyr was inco ⁇ orated toward the amino-terminus of the peptide. PTPase inhibitors were synthesized by similar methods with Fmoc-Pmp, see Marseigne et al., 1988, J. Org. Chem. 52:3621-3624, hereby inco ⁇ orated by reference. Phosphopeptides and phosphonopeptides were readily purified by HPLC, and gave the expected amino acid composition and FAB-MS values.
  • PTPase Substrates The major phosphorylation sites of the insulin receptor(IR), PDGF receptor (PDGF-F), pp60 c"src , the polyomavirus transforming protein, middle t, and putative phosphorylation sites of the endogenous insulin receptor substrate (IRS-1) were synthesized as phosphotyrosine peptides. Most of these sequences have only one Tyr residue. However, activation of the insulin receptor requires phosphorylation of three proximate Tyr residues (White et al., 1988, J.Biol. Chem. 2£2:2969) and all three monophosphopeptides were prepared.
  • the phosphopeptides were used as substrates of PTPase IB, a single catalytic domain PTPase with a side tissue distribution (Hunter et al., 1989, cell 58:1013).
  • Rat PTPase IB (Guan et al., 1990, Proc. Natl., Acad. Sci. USA £2:1501) was obtained by PCR and cloning the full-length cDNA into the bacterial expression vector, PKK233-2. Sequence specificity for peptide dephosphorylation was observed, with apparent K m values ranging from ⁇ 2 ⁇ M for the phospho-middle t sequence to >2 mM for pTyr itself (Table 3).
  • PTPase Inhibitors Representative phosphopeptide substrate sequences w prepared as phosphonopeptides for use as inhibitors. Pmp was synthesized chemically a racemic mixture which was not resolved prior to peptide synthesis. Synthetic product were, however, readily separated into two components by HPLC which corresponded t peptides having D- and L-Pmp, respectively.
  • Inhibitor Assays The activity of a peptide inhibitor can be assayed by determining the ability of the inhibitor to inhibit the dephosphorylation of a naturally occurring or synthetic substrate. These assays can be performed in cultured cells or in cell free systems e.g., by adding an inhibitor and following the phosphorylation of a predetermined component. Alternatively, purified or partially purified components can tested in vitro to determine the ability of an inhibitor to inhibit dephosphorylation by a specific PTPase, e.g., placental PTPase, Tonks et al., 1988 J. Biol. Chem 263:6731,
  • the substrate tested can be a naturally occurring substrate or a synthetic substrate.
  • Increasing the electronegativity of the phosphate moiety in an analog of phosphotyrosine increases the binding affinity of peptides containing the analog to a substrate such as protein containing an SH2 domain, and in some cases can exceed that of peptides containing phosphotyrosine.
  • the electronegativity of the phosphate moiety can be increased by substituting the hydrogens on the carbon between the phenyl ring and the phosphonate group with small, electronegative atoms such as fluorine or chlorine. Fluorine atoms are particularly desirable because they increase the electronegativity of the phosphate moiety without significantly adding to the bulk of the molecule.
  • Hydrolysis resistant phosphotyrosine analogs such as monofluorophosphonomethylphenylalanine and difluorophosphonomethylphenylalanine can be used in peptides and methods of the invention, e.g., in place of Pmp.
  • phosphotyrosine is indicated by filled squares ( ⁇ ).
  • Phosphotyrosine ( ⁇ ) was replaced in the peptide by Pmp (D), monofluorophosphonomethylphenylalanine (FPmp) (0), F2Pmp (star), hydroxyfluorophosphonomethylphenylalanine (HPmp) (O), and tyrosine (Tyr) ( ⁇ ).
  • Th ED50 values were 0.17 ⁇ 0.02 ⁇ M for pTyr, 1.O ⁇ O.1 ⁇ M for Pmp, 0.50 ⁇ 0.03 ⁇ M for FPm 0.17 ⁇ 0.02 ⁇ M for F2Pmp, 3.3 ⁇ 0.5 ⁇ M for HPmp, and >l,000 ⁇ M for Tyr.
  • Fig. 3 is a depiction of: panel A, the binding affinity of the peptide DXVPML (SEQ ID NO:46) for the SH2 domain of PI 3-kinase where X was pTyr (•) F2Pmp (O), the ED50 values were 0.15 ⁇ 0.03 ⁇ M pTyr and 0.17 ⁇ 0.02 ⁇ M for F2Pmp; panel B, the binding affinity of the peptide QXEEIP (SEQ ID NO:47) for the SH2 dom of Src where X was pTyr (•) or F2Pmp (O), the ED50 values were 5.7 ⁇ 0.7 ⁇ M for pTy and 1.0 ⁇ 0.2 ⁇ M for F2Pmp; panel C, the binding affinity of the peptide NXVNIE (SEQ ID NO: 48) for the SH2 region of Grb2 where X was pTyr (•), L-F2Pmp(0), or D- F2Pmp(D), the ED50 values were 0.9 ⁇
  • Fig. 3 indicates that in some cases F2Pmp can bind more poten to the SH2 domain of a protein than phosphotyrosine.
  • Panel B of Fig. 3 shows that Ac- F2Pmp-E-E-I-P-NH2 (SEQ ID NO: 47) binds more avidly to the SH2 domain of Src th phosphotyrosine.
  • F2Pmp peptides have been microinjected into living cells and have been effective in inhibiting ligand-stimulated effects.
  • the peptides of the invention may be administered to a mammal, particularly a human, on any suitable fashion, e.g., in one of the traditional modes (e.g. orally, parenterally, transdermally, or transmucosally), in a sustained release formulati 5 using a biodegradable biocompatible polymer, or by on-site delivery using micelles, ge and liposomes.
  • Dosages will vary, depending on factors such as, the disease being treate the half life of the substance, potency, route of administration, and the condition of the patient. l o Other embodiments are within the following claims.

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Abstract

Un peptide est capable d'inhiber l'interaction d'une protéine contenant un domaine SH2 avec une deuxième protéine contenant la séquence R1-méthionine-R2-métionine, dans laquelle R1 désigne phosphotyrosine ou un analogue de la phosphotyrosine ayant une fraction phosphoreuse résistante à l'hydrolyse et R2 désigne n'importe quel acide aminé.
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JP2008520575A (ja) * 2004-11-15 2008-06-19 セプタイア インコーポレイテッド プロテインチロシンホスファターゼ阻害剤およびその使用方法
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Title
J. BIOL. CHEM., Volume 267, Number 8, issued 15 March 1992, K.R. AUGER et al., "Polyoma Virus Middle T Antigen-pp60c-src Complex Associates with Purified Phosphatidylinositol 3-Kinase In Vitro", pp. 5408-5415. *
SCIENCE, Volume 250, No. 4983, issued 16 November 1990, D. ANDERSON et al., "Binding of SH2 Domains of Phospholipase Cgamma-1, GAP, and Src to Activate Growth Factor Receptors", pp. 979-982. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5861266A (en) * 1994-02-28 1999-01-19 New York University Treatment of diabetes mellitus and insulin receptor signal transduction
WO1995025118A3 (fr) * 1994-03-15 1995-11-16 Trustees Of Tufts University Inhibiteurs d'interactions des domaines sh2
WO1996030332A1 (fr) * 1995-03-31 1996-10-03 The Government Of The United States Of America, Represented By The Secretary, Department Of Healtha Nd Human Services Composes a base d'o-malonyltyrosyle, peptides contenant des composes a base d'o-malonyltyrosyle et leurs utilisations
EP0833629A4 (fr) * 1995-06-19 1998-09-16 Ontogen Corp Derives d'acide aryl-acrylique convenant comme inhibiteurs de proteine-tyrosine-phosphatase
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US6939952B2 (en) 1998-10-30 2005-09-06 Vanderbilt University Purified and isolated protein zero related (PZR) polypeptide
WO2003041729A1 (fr) * 2001-09-26 2003-05-22 Albert Einstein College Of Medicine Of Yeshiva University Inhibiteurs et ligands de ptp1b
EP1435989A4 (fr) * 2001-09-26 2006-05-03 Einstein Coll Med Inhibiteurs et ligands de ptp1b
AU2002363632B2 (en) * 2001-09-26 2007-05-24 Albert Einstein College Of Medicine Of Yeshiva University PTP1B inhibitors and ligands
US7736911B2 (en) 2004-04-15 2010-06-15 Albert Einstein College Of Medicine Of Yeshiva University Activity-based probes for protein tyrosine phosphatases
JP2008520575A (ja) * 2004-11-15 2008-06-19 セプタイア インコーポレイテッド プロテインチロシンホスファターゼ阻害剤およびその使用方法
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US7829737B2 (en) 2004-11-15 2010-11-09 Ceptyr, Inc. Protein tyrosine phosphatase inhibitors and methods of use thereof

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