WO1997015318A1 - Peptide inhibitors of a phosphotyrosine-binding domain containing protein - Google Patents
Peptide inhibitors of a phosphotyrosine-binding domain containing protein Download PDFInfo
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- WO1997015318A1 WO1997015318A1 PCT/US1996/017080 US9617080W WO9715318A1 WO 1997015318 A1 WO1997015318 A1 WO 1997015318A1 US 9617080 W US9617080 W US 9617080W WO 9715318 A1 WO9715318 A1 WO 9715318A1
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- 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
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Definitions
- the invention relates to peptides which interfere with the interaction of a phosphotyrosine-binding (PTB) domain containing protein with a PTB domain binding site; and, uses of the peptides.
- PTB phosphotyrosine-binding
- Shc is a member of a group of proteins that are collectively known as adaptor proteins. These adaptors, which are composed of protein-protein interaction domains such as the Src-homology 2 (SH2) and Src-homology 3 (SH3) domains, mediate protein-protein interactions that are important for signal transduction downstream of growth factor and cytokine receptors (Pawson, 1995). Shc has been shown to bind to a wide variety of activated growth factor and cytokine receptors.
- SH2 protein-protein interaction domains
- SH3 Src-homology 3 domains
- Shc was cloned from a human cDNA library in a screen for SH2 domain-containing proteins (Pelicci et al., 1992); Shc homologs in mouse (mShc) and drosophila (dShc) have also been cloned (Lai et al., 1995). Three proteins are encoded by the shc gene that differ from each other only in their amino-terminus (Lai et al., 1995; Pelicci et al., 1992).
- Shc results in cellular transformation of NTH3T3 fibroblasts and Ras-dependent neurite outgrowth of PC 12 cells, suggesting that Shc plays an important role in signal transduction leading to DNA synthesis and cell division or differentiation (Pelicci et al., 1992; Rozakis-Adcock et al., 1992).
- Shc contains an amino-terminal phosphotyrosine-binding (PTB) domain, a central Pro-rich region that contains the principal tyrosine phosphorylation site at Tyr 317, and an SH2 domain at its carboxy-terminus.
- PTB phosphotyrosine-binding domain
- the PTB domain which is highly conserved in Shc-related proteins, was recently identified based on its ability to bind to phosphotyrosine-containing proteins (Blaikie et al., 1994; Kavanaugh and Williams, 1994; van der Geer et al., 1995).
- SH2 domain recognizes phosphotyrosine present within the sequence Asn-Pro-X-P.Tyr and differs from SH2 domains that recognize phosphotyrosine in the context of carboxy-terminal residues (Kavanaugh et al., 1995; van der Geer et al., 1995).
- the Shc SH2 domain recognizes phosphotyrosine within the sequence P.Tyr-Glu/Leu/Ile/Tyr-X-Leu/Ile/ Met (Songyang et al., 1994).
- Shc becomes phosphorylated on tyrosine following stimulation with a wide variety of growth factors and cytokines (Burns et al., 1993; Crowe et al., 1994; Cutler et al., 1993; Lanfrancone et al., 1995; Pelicci et al., 1992; Pronk et al., 1993; Ravichandran et al., 1993;
- Tyrosine phosphorylation of Shc is essential for its interaction with the Grb2-Sos complex, which may provide a mechanism for Ras activation (Buday and Downward, 1993; Crowe et al., 1994; Egan et al., 1993; Gale et al., 1993; Li et al., 1993; Rozakis-Adcock et al., 1993; Rozakis-Adcock et al., 1992; Salcini et al., 1994). Shc has also been shown to bind physically to activated growth factor and cytokine receptors.
- the Shc PTB domain has been shown to bind to the activated nerve growth factor (NGF) receptor, the activated epidermal growth factor (EGF) receptor, polyoma middle T antigen, and to a 145 kDa protein that becomes phosphorylated on tyrosine in PDGF stimulated cells (Blaikie et al., 1994; Kavanaugh and Williams, 1994; van der Geer et al., 1995).
- the NGF receptor contains a single Shc-binding site at Tyr 490 that is present within a Asn-Pro-X-Tyr motif (Obermeier et al., 1994; Stephens et al., 1994).
- NGF receptors that have been mutated at Tyr 490 lack the ability to interact with Shc in vivo or with the PTB domain in vitro (Stephens et al., 1994).
- the present inventors have identified the residues within the Asn-Pro-X-P Tyr motif of phosphotyrosine-containing proteins (e.g. growth activated growth factors and cytokine receptors) that mediate the binding of the proteins to signalling proteins containing PTB domains.
- the present inventors found that the Asn and the phosphotyrosine residues within the Asn-Pro-X-P.Tyr motif of the phosphotyrosine-containing proteins mediate their binding to the PTB domain of Shc.
- an aliphatic residue that is five or six residues amino-terminal to the phosphotyrosine is required for binding.
- This aliphatic residue is missing from the insulin receptor autophosphorylation site which is unable to form a stable complex with Shc.
- the present inventors also analyzed the Shc PTB domain by in vitro mutagenesis and an evolutionarily conserved Arg residue was identified that is important for PTB binding to its ligands.
- the present invention relates to a peptide of the formula I
- X 1 represents Lys, Arg, His, Ser, Thr, Tyr, Asn, Leu, Val, or Glu
- a 1 represents Trp, Leu, Ala, Ser, Ile, Glu, Met, Gly, Cys, Phe, Pro, or Val
- a 2 represents Ala, Val, Leu, Ile, Ser, Met, Phe, Gly, Cys, Trp, or Pro
- X 2 represents Glu, Asn, Tyr, Thr, Ser, Asp, or Ile
- X 3 represents Pro, Met, Trp, Phe, Ala, Lys, Val, Leu, Ile, Gly, or Cys
- X 4 represents Leu, Ala, Glu, Gln, Asp, Asn, Tyr, Thr, or Ser
- X 5 represents P
- a peptide of the formula I is provided.
- X 1 represents His, Ser, Thr, Tyr, Asn, Leu, Val, or Glu
- X 2 represents Glu, Ser, Asp, or Ile
- X 3 represents Pro or Lys
- X 4 represents Leu, Ala, Glu, Gln, Asn, or Thr
- X 5 represents Phe, Leu, Ile, Gly, Arg, or Ser
- X 6 represents Ser, Thr, Met, Ala, Leu, Val, or Gly
- X 7 represents Asp, Ala, Val, Leu, Met, Ser, or Asn
- X 8 which may be present or absent, represents Leu, Ala, Gly, Asp, Ser, or Arg
- a 1
- a 2 represents Ala, Val, Leu, Ile, Ser, Met, or Phe, which interferes with the interaction of a PTB domain containing protein with a PTB domain binding site.
- X 1 represents Lys, Arg, His, preferably His
- X 2 represents Glu, Asn, Tyr, Thr, Ser, preferably Glu
- X 3 represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, preferably Pro
- X 4 represents Gln, Asp, Asn, Tyr, Thr, Ser, preferably Gln
- X 5 represents Phe, Trp,
- a 1 and A 2 represents Ile and the other of A 1 and A 2 represents Ile or Ala, preferably A 1 represents Ala and A 2 represents Ile, which interferes with the interaction of a PTB domain containing protein with a PTB domain binding site.
- X 1 represents Ser, Thr, Tyr, Asn or Glu, preferably Tyr
- X 2 represents Glu, Asn, Tyr, Thr
- Ser preferably Ser
- X 3 represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, preferably Pro
- X 4 represents Glu, Asp, preferably Glu
- X 5 represents Phe, Trp, Pro, Leu
- the invention also relates to truncations and analogs of the peptides of the invention.
- the invention also relates to the use of a peptide of the formula I or Ia to interfere with the interaction of a PTB domain containing protein with a PTB domain binding site; and, pharmaceutical compositions for inhibiting the interaction of a PTB domain containing protein with a PTB domain binding site.
- the invention relates to a method of modulating the interaction of a PTB domain containing protein with a PTB domain binding site comprising changing the amino acid Arg at position 175 in the PTB domain containing protein.
- the invention still further relates to a method for modulating the interaction of an insulin receptor with insulin receptor substrate 1 (IRS-1) or Shc comprising incorporating a large aliphatic amino acid at amino acids -5 or -6 amino terminal to the P.Tyr in the motif Asn-Pro-X-P.Tyr in the PTB domain of the insulin receptor.
- IRS-1 insulin receptor substrate 1
- Shc comprising incorporating a large aliphatic amino acid at amino acids -5 or -6 amino terminal to the P.Tyr in the motif Asn-Pro-X-P.Tyr in the PTB domain of the insulin receptor.
- Figure 1 A is an immunoblot showing P.Tyr-containing proteins bound to GST (lane 1)
- Figure IB is the immunoblot shown in Figure 1A stripped and reprobed with an antiserum raised against the NGF receptor;
- Figure 2 is a graph showing the results of surface plasmon resonance technology testing the ability of Wt and mutant phosphopeptides, based on the sequence around Tyr 490 the Shc-binding site in the NGF receptor to compete for binding of the GST-Shc PTB domain fusion protein to the immobilized polyoma middle T antigen peptide;
- Figure 3 is a schematic diagram showing the presence of an Asn-Pro-X-P.Tyr motif in the juxta membrane domains of the NGF and insulin receptors;
- Figure 4A is an immunoblot showing anti-Shc immunoprecipitates (lanes 1, 2, 5, 6, 9, and 10) from control (lanes 1, 5, and 9) and growth factor-stimulated (lanes 2, 6, and 10) NIH3T3 fibroblasts expressing Wt (lanes 1 and 2; NGFR) or Phe 490 mutant (lanes 5 and 6; F490NGFR) NGF receptors, or CHO cells expressing Wt insulin receptors (lanes 9 and 10; IR) analyzed by anti-P.Tyr immunoblotting; anti-NGF receptor (lanes 3, 4, 7, and 8) and anti-insulin receptor immuno-precipitates (lanes 11 and 12) from control (lanes 3, 7, and 11) and growth factor stimulated (lanes 4, 8, and 12) were analyzed in parallel;
- Figure 4B is an immunoblot showing Wt (lanes 1 and 2) and Phe 490 mutant (5 and 6) NGF receptors present in lysates from control (lanes 1 and 5) and NGF-stimulated (lanes 2 and 6) cells expressing Wt (NGFR) or Phe 490 mutant (F490NGFR) and insulin receptors (LR) present in lysates from control (lane 9) and insulin-stimulated (lane 10) cells incubated with GST-Shc PTB fusion proteins bound to glutathione-agarose, bound proteins were analyzed by anti-P.Tyr blotting;
- Figure 5A is an immunoblot showing GST-Shc PTB domain fusion proteins bound to glutathione-agarose after incubation with activated NGF receptors present in lysates of NGF-stimulated cells in the absence (lane 1) or presence (lanes 2-7) of 2 ⁇ M competing Wt and mutant phosphotyrosine containing peptides based on the sequence around Tyr 490, the Shc PTB domain binding site in the NGF receptor (lanes 2-5) or Tyr 960 an autophosphorylation site present within an Asn-Pro-X-P.Tyr motif in the insulin receptor (lanes 6 and 7);
- Figure 5B is a graph showing the results of testing phosphopeptides based on the sequence around Tyr 490, the Shc-binding site in the NGF receptor (H-I-I-E-N-P-Q-p. Y-F- S-D; ( ⁇ ) or Tyr.
- Figure 6A is an immunoblot showing GST fusion proteins containing Wt (lanes 1 and 2) or mutant (lanes 3-11) Shc PTB domains after incubation with NGF receptors present in lysates of control (lane 1) and NGF-stimulated cells (lanes 2-11), bound proteins were analyzed by anti-P.Tyr blotting,
- Figure 6B is an immunoblot showing human EGF receptors bound to GST fusion proteins containing Wt (lanes 1 and 2) or Met 175 (lane 3) and Lys 175 (lane 4) mutant human Shc PTB domains in lysates from control (lane 1) or EGF -stimulated cells (lanes 2- 4) analyzed by anti-P.Tyr blotting, and in parallel GST (lane 8) and GST fusion proteins containing Wt (lane 7) or an Ala 151 mutant (lane 9) drosophila Shc PTB domain bound to glutathione-agarose, incubated with fly lysates containing activated Torso-DER chimeric proteins that contain the cytoplasmic domain of DER; bound proteins were detected by anti-P.Tyr blotting;
- Figure 7 shows the amino acid sequences of PTB binding domains of mammalian and Drosophila Shc homologues
- Figure 8 are immunoblots showing competitive inhibition of EGF receptor binding to GST-ShcB analyzed by anti-phospho-tyrosine antibody
- Figure 9 is an immunoblot showing competitive inhibition of EGF receptor binding to GST-ShcB analyzed by anti-phospho-tyrosine antibody
- Figure 10 are immunoblots showing a dose-response analysis in a competitive inhibition assay of EGF receptor binding to GST-ShcB analyzed by anti-phospho-tyrosine antibody;
- Figure 11 are bar graphs showing proliferation of HER14 cells treated with peptides of the invention.
- Figure 12 is a bar graph showing proliferation of HER14 cells treated with peptides of the invention.
- Figure 13 is a bar graph showing proliferation of HER14 cells treated with cyclic peptides of the invention.
- Figure 14 are bar graphs showing proliferation of SupM2 cells treated with peptides of the invention.
- Figure 15 are immunoblots showing MAPK activation on PC 12 cells treated with peptides of the invention.
- Figure 16 are immunoblots showing activated MAPK on PC 12 cells treated with peptides of the invention.
- A Ala - alanine
- C Cys - cysteine
- D Asp- aspartic acid
- E Glu - glutamic acid
- F Phe - phenylalanine
- G Gly - glycine
- H His - histidine
- I Ile - isoleucine
- K Lys - lysine
- L Leu - leucine
- M Met - methionine
- N Asn - asparagine
- P Pro - proline
- Q Gln - glutamine
- R Arg - arginine
- S Ser - serine
- T Thr - threonine
- V Val - valine
- W Trp- tryptophan
- Y Tyr - tyrosine
- p.Y. P.Tyr - phosphotyrosine.
- the present invention relates to a peptide of the formula I X 1 - A 1 - A 2 - X 2 - Asn - X 3 - X 4 - P.Tyr - X 5 - X 6 - X 7 -X 8 I wherein X 1 represents Lys, Arg, His, Ser, Thr, Tyr, Asn, Leu, Val, or Glu, A 1 represents Trp, Leu, Ala, Ser, Ile, Glu, Met, Gly, Cys, Phe, Pro, or Val, and A 2 represents Ala, Val, Leu, Ile, Ser, Met, Phe, Gly, Cys, Trp, or Pro, X 2 represents Glu, Asn, Tyr, Thr, Ser, Asp, or Ile, X 3 represents Pro, Met, Trp, Phe, Ala, Lys, Val, Leu, Ile, Gly, or Cys, X 4 represents Leu, Ala, Glu, Gln,
- X 7 represents Asp, Ala, Val, Leu, Met, Ser, or Asn
- X 8 which may be present or absent, represents Leu, Ala, Gly, Asp, Ser, or Arg
- a 1 represents Trp, Leu, Ala, Ser, Ile
- a 2 represents Ala, Val, Leu, Ile, Ser, Met, or Phe, which interferes with the interaction of a PTB domain containing protein with a PTB domain binding site.
- X 1 represents Lys, Arg, His, preferably His
- X 2 represents Glu, Asn, Tyr, Thr, Ser, preferably Glu
- X 3 represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, preferably Pro
- X 4 represents Gln, Asp, Asn, Tyr, Thr, Ser, preferably Gln
- X 5 represents Phe, Trp, Pro, Leu, Ala, Val, Ile, Gly, Cys, Met, preferably Phe
- X 6 represents Ser, Thr, Tyr, Asn, Glu, preferably Ser
- X 7 represents Asp, Glu, preferably Asp
- one of A 1 and A 2 represents Ile and the other of A 1 and A 2 represents Ile or Ala
- a peptide of the formula Ia is provided X 1 - A 1 - A 2 - X 2 - Asn - X 3 - X 4 - P.Tyr - X 5 - X 6 - X 7 Ia wherein X 1 represents Ser, Thr, Tyr, Asn or Glu, preferably Tyr, X 2 represents Glu, Asn, Tyr, Thr, Ser, preferably Ser, X 3 represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, preferably Pro, X 4 represents Glu, Asp, preferably Glu, X 5 represents Phe, Trp, Pro, Leu, Ala, Val, Ile, Gly, Cys, Met preferably Leu, X 6 represents Ser, Thr, Tyr, Asn, Glu, preferably Ser, X 7 represents Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro,
- Preferred peptides of the invention include the following His-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp, His-Ala-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp, His-Ile-Ala-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp, Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp-Ala, Tyr-Ala-Ile-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-Ala, Thr-Trp-Ile-Glu-Asn-Lys-Leu-P.Tyr-Gly-Met-Ser-Asp, Thr-Trp-Ile-Glu-Asn-Lys-Leu
- Truncated peptides may comprise peptides of about 7 to 10 amino acid residues
- the truncated peptide has the sequence A 2 -X 2 -Asn-X 3 -X 4 -P Tyr or A 2 -X 2 -Asn-X 3 -X 4 -P Tyr-X 5 wherein A 2 , X 2 , X 3 , X 4 , and X 5 are as defined above
- the truncated peptide has the sequence Leu/Ile-X 2 -Asn-Pro-X 4 -P Tyr, wherein X 2 represents Glu, Ser, Asp, or Ile, and X 4 represents Leu, Ala, Glu, Gln, Asn, or Thr
- the truncated peptides may have an ammo group (-NH 2 ), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end
- the truncated peptides may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end
- the peptides of the invention may also include analogs of the peptide of the Formula I, and/or truncations of the peptide, which may include, but are not limited to the peptide of the formula I containing one or more amino acid insertions, additions, or deletions, or both.
- Analogs of the peptide of the invention exhibit the activity characteristic of the peptide i.e. interference wim the interaction of a PTB domain containing protein with a PTB domain binding site, and may further possess additional advantageous features such as increased bioavailability, stability, or reduced host immune recognition.
- amino acid insertions may be introduced into a peptide of the formula I preferably outside the sequence A 2 -X 2 -Asn-X 3 -X 4 -P.Tyr-X 5 .
- amino acid insertions may be made between X 1 and A 1 or between X 5 and X 6 , or X 6 and X 7 .
- Amino acid insertions may consist of a single amino acid residue or sequential amino acids.
- One or more amino acids may be added to the right or left termini of a peptide of the invention.
- Examples of such analogs include Ala-Leu-Leu-Leu-Ser-Asn-Pro-Ala-P.Tyr.-Arg-Leu-Leu-Leu-Ala; Gly-Pro-Leu-Tyr-Ala-Ser-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-Ala-Ser-Asp-Val-Phe; Pro-Val-Ser-Val-As ⁇ -Asn-Pro-Glu-P.Tyr-Leu-Leu-Asn-Ala-Gln-Lys; Leu-Ser-Leu-Leu-Ser-Asn-Pro-Thr-P.Tyr-Ser-Val-Met-Arg-Ser-Lys; Val-Ser-Ser-Leu-Asn-Glu-Met-Ile
- Deletions may consist of the removal of one or more amino acids, or discrete portions from the peptide sequence preferably outside the A 2 -X 2 -Asn-X 3 -X 4 -P.Tyr sequence.
- the deleted amino acids may or may not be contiguous.
- the lower limit length of the resulting analog with a deletion mutation is about 7 amino acids.
- Cyclic derivatives of the peptides of the invention are also part of the present invention. Cyclization may allow the peptide to assume a more favorable conformation for association with a PTB domain containing protein. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The side chains of P.Tyr and Asn may be linked to form cyclic peptides. The components that form the bonds may be side chains of amino acids, non-amino acid components or a combination of the two.
- Preferred cyclic peptides of the invention include cyclo-(Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Pro-Gly, and cyclo-(Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp-Ala-Pro- Gly) (SEQ. ID. NOs.
- cyclic peptides are contemplated that have a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.
- An example of such a cyclic peptide is a peptide of the invention with an Ile in the left position (i.e. a terminal A 1 or A 2 is Ile) and the amino acids Pro-Gly at the right position.
- the amino group of the Ile and the carboxyl group of the Gly form a peptide bond resulting in a cyclic peptide.
- the 3D structure of the cyclic peptide is similar to the original structure of the PTB binding site of TrkA.
- cyclic peptide that has a beta-turn in the right position: cyclo-(Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Pro-Gly) (SEQ. ID. NO. 26 in the Sequence Listing).
- Asn-Pro-Gln-P.Tyr take a native beta-turn
- Ser-Pro-Gly-Ile make another beta-turn on the other side
- the central part adopts an antiparallel beta-sheet.
- a beta-sheet has two faces, and the peptide binds to the PTB domain with the face on which the side chains of Ile, Asn, and P.Tyr extend.
- the side chains of Glu and Phe are on the other face, and may not affect the binding affinity. It may be possible to control the binding specificity by the side-chain of Gln as this side chain may contact the PTB domain.
- a more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines.
- the two cysteines are arranged so as not to deform the beta-sheet and turn.
- the peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion.
- the relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations
- the invention also includes a peptide conjugated with a selected peptide, protein, or a selectable marker (see below) to produce fusion proteins.
- a peptide of the invention may be conjugated with a peptide which facilitates entry into cells.
- the peptides of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.
- inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc.
- organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulf
- nucleic acid molecules which encode a peptide of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the peptide.
- Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses so long as the vector is compatible with the host cell used.
- the expression vectors contain a nucleic acid molecule encoding a peptide of the invention and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence.
- Suitable regulatory sequences may be obtained from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may also be incorporated into the expression vector.
- the recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells.
- Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG.
- the selectable markers may be introduced on a separate vector from the nucleic acid of interest.
- the recombinant expression vectors may also contain genes which encode a fusion portion which provides increased expression of the recombinant peptide; increased solubility of the recombinant peptide; and/or aid in the purification of the recombinant peptide by acting as a ligand in affinity purification.
- a proteolytic cleavage site may be inserted in the recombinant peptide to allow separation of the recombinant peptide from the fusion portion after purification of the fusion protein.
- fusion expression vectors examples include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
- GST glutathione S-transferase
- Recombinant expression vectors may be introduced into host cells to produce a transformant host cell.
- Transformant host cells include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention.
- transfection are intended to include the introduction of nucleic acid (e.g. a vector) into a cell by one of many techniques known in the art.
- nucleic acid e.g. a vector
- prokaryotic cells can be transformed with nucleic acid by electroporation or calcium-chloride mediated transformation.
- Nucleic acid can be introduced into mammalian cells using conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells may be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
- Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
- the peptides of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells.
- Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).
- the peptides of the invention may be tyrosine phosphorylated using the method described in Reedijk et al. (The EMBO Journal 11(4): 1365, 1992).
- tyrosine phosphorylation may be induced by infecting bacteria harbouring a plasmid containing a nucleotide sequence encoding a peptide of the invention, with a ⁇ gtl 1 bacteriophage encoding the cytoplasmic domain of the Elk tyrosine kinase as a LacZ-Elk fusion.
- Bacteria containing the plasmid and bacteriophage as a lysogen are isolated. Following induction of the lysogen, the expressed peptide becomes phosphorylated by the Elk tyrosine kinase.
- the peptides of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis
- the peptides may be synthesized using 9-fluorenyl methoxycarbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphotyrosine as the N-fluorenylmethoxy-carbonyl-O-dimethyl phosphono-L-tyrosine derivative.
- Fmoc 9-fluorenyl methoxycarbonyl
- N-terminal or C-terminal fusion proteins comprising a peptide of the invention conjugated with other molecules may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the peptide, and the sequence of a selected protein or selectable marker with a desired biological function.
- the resultant fusion proteins contain the peptide fused to the selected protein or marker protein as described herein.
- proteins which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.
- the peptides of the invention may be used to prepare monoclonal or polyclonal antibodies. Conventional methods can be used to prepare the antibodies. As to the details relating to the preparation of monoclonal antibodies reference can be made to Goding, J.W., Monoclonal Antibodies: Principles and Practice, 2nd Ed., Academic Press, London, 1986. As discussed below, the antibodies may be used to identify proteins with PTB domain binding sites.
- the peptides and antibodies specific for the peptides of the invention may be labelled using conventional methods with various enzymes, fluorescent materials, luminescent materials and radioactive materials. Suitable enzymes, fluorescent materials, luminescent materials, and radioactive material are well known to the skilled artisan. Labeled antibodies specific for the peptides of the invention may be used to screen for proteins with PTB domain binding sites, and labeled peptides of the invention may be used to screen for PTB domain containing proteins such as Shc.
- PTB domain containing protein refers to a protein or peptide which comprises or consists of a PTB domain.
- a PTB domain is a region which is a domain of -160 amino acids which was originally identified in Shc and Sck (Kavanaugh, V.M. Et al., 1995 Science, 268.1177-1179; Bork, RP, and Margolis, B, Cell, Vol 80:693-694, 1995; Craparo, A., et al., 1995, J. Biol. Chem.
- the PTB domain comprises residues 46 to 208 in the 52 kDa isoform of Shc.
- the sequences of several known PTB domains are aligned in Figure 7. In Figure 7, residues that are conserved within the sequences are shaded.
- PTB domain containing proteins are mammalian Shc and Sck, IRS-1, and homologues of Shc including Drosophila Shc, and mouse Shc. Other proteins that contain homologous PTB domains have been identified using data base search methods (Bork, RP, and Margolis, B. Cell, Vol 80:693-694, 1995). PTB domain containing proteins may also be identified by screening a cDNA expression library with a protein containing a sequence with high affinity to PTB domains, i.e. a PTB domain binding sequence or a peptide of the invention which may be labeled. PTB domain containing proteins may also be screened using antibodies specific for the PTB domain.
- a PTB domain that binds to the consensus sequence Leu/Ile-X- Asn-Pro-X-P.Tyr found in growth factors may be identified by screening a cDNA expression library with proteins based on the consensus sequence. PCR (Wilks, A.F., Proc. Natl. Acad. Sci. U.S.A. Vol. 86, pp. 1603-1607, March 1989) or low stringency screening (Hanks, S.K., Proc. Natl. Acad. Sci. U.S.A. Vol. 84, pp 388-392, January 1987) with the PTB domain specific probe can be used.
- the term " PTB domain binding site" refers to a sequence with high affinity to PTB domains.
- PTB domain binding sequences have been identified in activated growth factors such as activated nerve growth factor receptor, activated epidermal growth factor (EGF) receptor, polyoma middle T antigen, and SHIP (Blaikie et al., 1994; Kavanaugh and Williams, 1994; van der Geer et al., 1995; Scott et al., 1996), ErbB2, ErbB3, TrkA, TrkB, TrkC, MCKlOb, insulin receptor, IGF-1 receptor, and IL-4 receptor.
- PTB domain binding sites may be identified by screening with PTB domain containing proteins or with antibodies specific for the peptides of the invention.
- the phrase "interfere with the interaction of” refers to the ability of the peptides of the invention to inhibit the binding of a PTB domain containing protein to a PTB domain binding site thereby affecting regulatory pathways that control gene expression, cell division, cytoskeletal architecture and cell metabolism.
- regulatory pathways are the Ras pathway, the pathway that regulates the breakdown of polyphosphoinositides through phospholipase C, and PI-3-kinase activated pathways, such as the rapamycin-sensitive protein kinase B (PKB/Akt) pathway.
- the peptides of the invention have been specifically shown to interfere with the interaction of the PTB domain of Shc and phosphotyrosine-containing peptides based on the sequence around Tyr 490 in activated nerve growth factor receptor and based on the Shc binding site in polyoma middle T antigen. Accordingly, the activity of a peptide of the invention may be confirmed by assaying for the ability of the peptide to interfere with the interaction of the PTB domain of Shc and phosphotyrosine-containing peptides based on the sequence around Tyr 490 in activated nerve growth factor receptor, or based on the Shc binding site in polyoma middle T antigen.
- Computer modelling techniques known in the art may also be used to observe the interaction of a peptide of the invention, and truncations and analogs thereof with a PTB domain containing protein (for example, Homology Insight II and Discovery available from BioSym/Molecular Simulations, San Diego, California, U.S.A.). If computer modelling indicates a strong interaction, the peptide can be synthesized and tested for its ability to interfere with the binding of the PTB domain of Shc and phosphotyrosine-containing peptides as discussed above.
- the peptides of the invention mediate the interactions of PTB domain containing proteins with PTB domain binding sites on proteins such as growth factors and cytokine receptors which regulate pathways that control gene expression, cell division, cytoskeletal architecture and cell metabolism.
- the peptides may therefore be used in the treatment of conditions involving perturbation of such regulatory pathways.
- the peptides may be useful in treating disorders involving excessive proliferation including various forms of cancer such as leukemias, lymphomas (Hodgkins and non-Hodgkins), sarcomas, melanomas, adenomas, carcinomas of solid tissue, hypoxic tumors, squamous cell carcinomas of the mouth, throat, larynx, and lung, genitourinary cancers such as cervical and bladder cancer, hematopoietic cancers, head and neck cancers, and nervous system cancers, ovarian cancer, breast cancer, glioblastoma, benign lesions such as papillomas, arthrosclerosis, angiogenesis, and viral infections, in particular HIV infections; and autoimmune diseases including systemic lupus erythematosus, Wegener's granulomatosis, rheumatoid arthritis, sarcoidosis, polyarthritis, pemphigus, pemphigoid, erythema multiforme, S
- the invention also relates to a pharmaceutical composition
- a pharmaceutical composition comprising a peptide of the invention for use as an antagonist of the interaction of a PTB domain containing protein, preferably Shc and a PTB domain binding site, preferably an activated growth factor or cytokine receptor.
- the peptides of the invention may be formulated into pharmaceutical compositions for adminstration to subjects in a therapeutically active amount and in a biologically compatible form suitable for administration in vivo i.e. a form of the peptides to be administered in which any toxic effects are outweighed by the therapeutic effects.
- the peptides may be administered to living organisms including humans, and animals.
- a therapeutically active amount of the pharmaceutical compositions of the invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
- a therapeutically active amount of a peptide may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regime may be adjusted to provide the optimum therapeutic response.
- the peptides may be administered in a convenient manner such as by injection
- the peptides may be coated in a material to protect them from the action of enzymes.
- the peptides may also be used in combination with organic substances for prolongation of their pharmacologic actions. Examples of such organic substances are non-antigenic gelatin, carboxymethylcellulose, sulfonate or phosphate ester of alginic acid, dextran, polyethylene glycol and other glycols, phytic acid, polyglutamic acid, and protamine.
- compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of a peptide is combined in a mixture with a pharmaceutically acceptable vehicle.
- Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
- the compositions include, albeit not exclusively, solutions of the peptides in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
- the peptides may also be incorporated in liposomes or similar delivery vehicles.
- peptides and compositions of the invention may be confirmed in in vitro cell penetration assays.
- the effects of the peptides upon cellular functions in vivo may be confirmed using electroporation techniques (See Raptis, L., and
- the utility of the peptides and compositions of the invention may also be confirmed in in vivo animal experimental model systems.
- therapeutic utility in proliferative disorders may be tested by examining the ability of a substance to suppress the growth of a transplantable tumor.
- Particular in vivo animal models which may be used include the growth of human tumor cell lines (e.g. glioblastomas) in nude mice; and the development of tumors in mice that carry MMTV-polyomavirus middle T antigen or MMTV-neu transgenes, which result in the development of mammary carcinoma.
- CHO cells expressing Wt insulin receptors (White etal., 1988) were grown in F12 medium containing 25 mM Hepes pH 7.4, and 10% fetal bovine serum.
- NIH3T3 cells expressing Wt and Phe 490 mutant NGF receptor (Stephens et al., 1994) were grown Dulbecco-Vogt's modified Eagle medium (DMEM) containing 10% calf serum (CS).
- DMEM Dulbecco-Vogt's modified Eagle medium
- CS calf serum
- NIH3T3 cells overexpressing the human EGF receptor (Honegger et al., 1987) were grown in DMEM containing 10% CS and 400 ⁇ g/ml G418.
- the monoclonal anti-insulin receptor antibody 51 was obtained from Dr. I. Goldfine
- Control and growth factor stimulated cells were rinsed twice with cold PBS and lysed in 1 ml 50 mM Hepes pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X100, 1.5 mM MgCl 2 , 1 mM EGTA, 100 mM NaF, 10 mM Sodium Pyrophosphate, 500 ⁇ M Sodium Vanadate, 1 mM PMSF, 10 ⁇ g/ml Aprotinin, and 10 ⁇ g/ml Leupeptin (PLC-lysis buffer) per 10 cm dish. Immunoprecipitations and PTB-binding assays in the absence or presence of 2 or 5 ⁇ M competing phosphopeptide were performed exactly as described previously (van der Geer et al., 1995).
- the surface was regenerated using 2 M Guanidinium-HCl.
- Torso-DER in transgenic flies.
- Transgenic flies expressing the activated Torso-DER chimeric protein expressed under the control of the heat shock promoter were obtained and protein expression was induced by growing the flies at 37°C for 45 min after which they were allowed to recover at room temperature for 2.5 hr. Lysates were made as described before (Lai et al., 1995).
- the PTB domain was found to bind tyrosine phosphorylated proteins that contain phosphorylation sites present within the sequence Asn-Pro-X-P.Tyr. To confirm that it is indeed the Asn-Pro-X-P.Tyr motif that is recognized by the PTB domain, it was shown that peptides that contain a phosphotyrosine within the sequence Asn-Pro-X-P.Tyr can compete for binding of the Shc PTB domain to activated growth factor receptors. The specificity was confirmed by sequencing peptides present in a degenerate phosphopeptide library that bind to the Shc PTB domain (Songyang etal., 1995).
- the Wt phosphopeptide (His-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp) competed efficiently for binding. Changing the Asn at position -3 (relative to the P.Tyr) to Ala completely abolished binding, whereas changing the Pro at -2 to Ala reduced the affinity of the PTB-peptide interaction.
- the identity of the NGF receptor was confirmed by stripping and reprobing the blot with a polyclonal antiserum raised against the NGF receptor (Figure 1B).
- the insulin receptor which contains a bona fide autophosphorylation site that is present within the sequence Asn-Pro-Glu-P.Tyr, lacks the ability to bind to Shc (Kovacina and Roth, 1993; Pronk et al., 1993). Tyr 960 in the insulin receptor is present in the juxta membrane domain, between the membrane and the kinase domain, in a position very similar to Tyr 490 in the NGF receptor (see Figure 3). The inability of the insulin receptor to associate stably with Shc was confirmed in coimmunoprecipitation experiments in which Shc immunoprecipitates were analyzed for associated proteins by anti-P.Tyr immunob lotting ( Figure 4A).
- Wt but not Phe 490 mutant NGF receptors can be detected in Shc immunoprecipitates from NGF-stimulated cells.
- insulin receptors were absent from Shc immunoprecipitates from insulin stimulated CHO cells overexpressing the Wt insulin receptor (CHO-IR cells).
- CHO-IR cells Wt insulin receptor
- GST fusion proteins containing the Shc PTB domain were incubated with lysates of control and insulin-stimulated CHO-IR cells and bound proteins were visualized by anti- P.Tyr immunoblotting.
- Wt and Phe 490 NGF receptors were included as controls.
- the NGF receptor and several other proteins with well defined Shc-binding sites often contain large aliphatic residues at six and five residues amino-terminal of the phosphorylated Tyr residue. These large aliphatic residues are absent from the insulin receptor, which has an Ala and a Ser six and five residues amino-terminal to Tyr 960 (Table 1). To test the possibility that these residues are important for PTB binding, several substitutions at these positions were made in the NGF receptor peptide and mutant peptides were tested for their ability to block binding of the PTB domain to the NGF receptor and to the polyoma middle T antigen phosphopeptide.
- Shc binding to activated growth factor receptors appears to be an important step in the initiation of signal transduction towards DNA synthesis and cell division or differentiation.
- Shc binding sites are particularly well characterized in the NGF receptor and in polyoma middle T antigen.
- the NGF receptor Shc binds to Tyr 490 in the juxta membrane domain ( Figure 3). Mutation of Tyr 490, in addition to mutation of the PLC ⁇ - binding site, completely blocks NGF-induced neuronal differentiation in PC 12 cells (Stephens et al., 1994). Mutation of Tyr 250, which is the Shc binding site, in polyoma middle T antigen blocks cellular transformation (Campbell et al., 1994; Dilworth et al., 1994). The EGF receptor also interacts strongly with Shc, although the precise contribution of different autophosphorylation sites in the EGF receptor carboxy-terminus remains unresolved (Batzer et al., 1994; Okabayashi et al., 1994).
- the PTB domain at the amino-terminus of Shc may be the important mediator of Shc-growth factor receptor interactions.
- Asn-Pro-X-P.Tyr motifs are conserved in a large number of Shc binding proteins and Asn-Pro-X-P.Tyr-containing peptides compete efficiently for Shc PTB binding to activated growth factor receptors, such as the receptors for EGF and NGF (Blaikie et al., 1994; Campbell et al., 1994; Kavanaugh et al., 1995; van der Geer and Pawson, 1995; van der Geer et al., 1995).
- EGF epidermal growth factor receptor
- the activated insulin receptor which also has an autophosphorylation site contained within an Asn-Pro-X-Tyr motif, does not bind stably to Shc in vivo or in vitro (Kovacina and Roth, 1993; Pronk et al., 1993).
- Shc becomes phosphorylated in response to insulin and the Shc PTB domain was shown to interact with Tyr 960 in the insulin receptor using the two-hybrid method in yeast (Gustafson et al., 1995).
- the present inventors have shown that the presence of an aliphatic residue five or six residue amino-terminal to the P.Tyr is important for high affinity binding by the Shc PTB domain.
- a phosphopeptide with two Ala residues at these positions still binds to the Shc PTB domain but with an affinity that is approximately three fold lower than that for binding of a phosphopeptide with an Ile at either position -6 or -5 (Table 1).
- the presence of a Ser five residues amino-terminal to the P.Tyr disrupts high affinity binding completely.
- a peptide, derived from the insulin receptor, that lacked the ability to bind to the Shc PTB domain was changed into a PTB-binding site with a single amino acid substitution at a residue outside the Asn-Pro-X-P.Tyr motif.
- PTB-binding specificity enables accurate predictions to be made as to which proteins will bind to particular PTB-containing adaptor or signalling molecules.
- it enables manipulation of the repertoire of PTB domain-containing proteins that are recruited by growth factor receptors without changing the actual phosphate acceptor sites.
- phosphorylation of both the insulin receptor substrate 1 (LRS-1) and Shc appears to depend on a low affinity interaction with the insulin receptor at Tyr 960 (Backer et al., 1990; White et al., 1988; Yonezawa et al., 1994).
- Shc appears to be important for signal transduction downstream of growth factor and cytokine receptors (Burns et al., 1993; Crowe et al., 1994; Cutler et al., 1993; Lanfrancone et al., 1995; Pelicci et al., 1992; Pronk et al., 1993; Ravichandran et al., 1993; Segatto et al., 1993; Yokote et al., 1994).
- Shc may be involved in Ras activation presumably through its interaction with Grb2 and Sos (Buday and Downward, 1993; Crowe et al., 1994; Egan et al., 1993; Gale et al., 1993; Li et al., 1993; Myers et al., 1994; Rozakis-Adcock et al., 1993; Rozakis-Adcock et al., 1992; Salcini et al., 1994; Sasaoka et al., 1994).
- C refers to a cyclic peptide
- C-1,3,4,5 are cyclized by the amino- and carboxyl termini by an amide bond
- C-2 is cyclized by a disulfide bond between two cysteines on each of the N- and C-termini
- P refers to peptides which have penetrating sequences on the N-terminus where P-1 and P-2 are basic charged penetrating sequences with the latter having phosphorylated tyrosine residues;
- P-3 and P-4 have a hydrophobic penetrating sequence with the latter having phosphorylated tyrosine residues; and
- P-5" was obtained by coupling with penetratin 1 (Appligene) and CGHIIENPQPYFSD.
- GST-ShcB and GST-R175M fusion proteins were prepared as described in van der Geer et al., 1995.
- HER14 cells (3T3 cells expressing EGF-R) were starved in 0.5% CS media for 24 hours and stimulated with 100 ng/ml EGF for 5 min. Cells were lysed and mixed with GST, GST-ShcB or GST-R175M beads. Proteins which bound to beads were resolved on SDS-PAGE and detected by anti-phospho-Tyr antibody (4G10) or by anti-EGF-R .
- Peptide localization in cells To examine peptide localization, cells were treated with P-1 or P-2 peptide for 4 hours, stained with anti-phospho-Tyr and rhodamine-conjugated antibody, and observed with a confocal microscope. A Z-scan was carried out to make images in each 0.15 ⁇ m section from the top of the cells to the bottom. The image analysis of cell staining demonstrated that the P-2 peptide localized in the cytoplasm of cells, and not in the nucleus. Cells treated with P-1 peptide were not stained by anti-phospho-Tyr antibody, confirming the specifity of the immunofluoroscence staining.
- HER14 cells were starved for 24 hours (Figure 11, Panel a), or 48 hours ( Figure 11, Panel b), treated with P-1 or P-2 peptide for 2 hrs and stimulated with 100ng/ml EGF.
- Cell proliferation was measured by ⁇ -TdR uptake ( Figure 11).
- P-1 and P-2 peptides slightly inhibited the proliferation of cells compared to the positive control i.e. EGF alone (a).
- EGF alone a
- both peptides markedly prevented cell proliferation. In particular, about 84% inhibition was observed at 1.25 ⁇ M.
- Both the P-1 and P-2 peptide demonstrated inhibitory activity, suggesting a non-specific effect was induced by adding peptides.
- the effect may be due to the internal phosphorylation of P-1 peptide by activated kinase(s) after growth factor stimulation.
- the above experiments were repeated with cells which were serum starved for 24 hrs. Cells pretreated with P-1 or P-2 did not show any decrease of cell growth rate when compared to EGF treated cells. Cells pretreated with C-2 inhibited cell growth roughly in a dose dependent manner ( Figure 12). The experiment was repeated using C-1 peptide as a negative control, and C-2 did not inhibit cell growth ( Figure 13).
- SupM2 A non-Hodgkin's lymphoma cell line, SupM2 was used in cell proliferation assays as described above.
- SupM2 has a chromosomal translocation, resulting in the expression of a fusion protein of Alk and Npm.
- the Alk/Npm fusion protein has a motif which is expected to be a Shc PTB binding domain, and the cell proliferation of SupM2 is believed to be dependent on the Shc pathway.
- the application contains sequence listings which form part of the application.
- FIG. 1A and Figure 1B The Asn present within the Asn-Pro-X-P.Tyr motif is essential for binding to the PTB domain.
- Figure 1A GST (lane 2) and GST Shc PTB
- Wt NGF receptor His-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Asp (lane 4); NGF receptor Asn(-3)Ala (NGFR Ala -3) mutant: His-Ile-Ile-Glu-Ala-Pro-Gln-P.Tyr-Phe-Ser-Asp (lane 5); NGF receptor Pro(-2)Ala (NGFR Pro -2): His-Ile-Ile-Glu-Asn-Ala-Gln-P.Tyr-Phe-Ser-Asp (lane 6).
- Figure 1B The blot shown under A was stripped and reprobed with an antiserum raised against the NGF receptor.
- H-I-I-E-N-P-Q-P.Y-F-S-D (A); Ala -3, H-I-I-E-A- P-Q-P.Y-F-S-D:( o ); Ala -2, H-I-I-E-N-A-Q-P.Y-F-S-DP(•).
- FIG. 3 Presence of a Asn-Pro-X-P.Tyr motif in the juxta membrane domains of the NGF and insulin receptors. Both the NGF receptor and the insulin receptor contain an autophosphorylation site within an Asn-Pro-X-P.Tyr motif in the juxta membrane domain, between the membrane and the kinase domain. In both receptors the tyrosine residues within these motif become phosphorylated upon receptor activation, but in contrast to the NGF receptor, the insulin receptor lacks the ability to stably associate with Shc.
- FIG. 4A and Figure 4B The Shc PTB domain does not stably bind to the Asn-Pro-X-P.Tyr motif in the insulin receptor.
- Figure 4A Anti-Shc immunoprecipitates (lanes 1, 2, 5, 6, 9, and 10) from control (lanes 1, 5, and 9) and growth factor-stimulated (lanes 2,
- NIH3T3 fibroblasts expressing Wt (lanes 1 and 2; NGFR) or Phe 490 mutant (lanes 5 and 6; F490NGFR) NGF receptors, or CHO cells expressing Wt insulin receptors (lanes 9 and 10; IR) were analyzed by anti-P.Tyr immunoblotting.
- Anti-NGF receptor (lanes 3, 4, 7, and 8) and anti-insulin receptor immunoprecipitates (lanes 11 and 12) from control (lanes 3, 7, and 11) and growth factor stimulated (lanes 4, 8, and 12) were analyzed in parallel.
- Figure 4B Figure 4B.
- Wt (lanes 1 and 2) and Phe 490 mutant (5 and 6) NGF receptors present in lysates from control (lanes 1 and 5) and NGF-stimulated (lanes 2 and 6) cells expressing Wt (NGFR) or Phe 490 mutant (F490NGFR) and insulin receptors (IR) present in lysates from control (lane 9) and insulin-stimulated (lane 10) cells were incubated with GST-Shc PTB fusion proteins bound to glutathione-agarose. Bound proteins were analyzed by anti-P.Tyr immunoblotting.
- Anti-NGF receptor immunoprecipitates (lanes 3, 4, 7, and 8) and anti-insulin receptor immunoprecipitates (lanes 11 and 12) from control (lanes 3, 7, and 11) and growth factor-stimulated (lanes 4, 8, and 12) cells were analyzed in parallel.
- Figure 5A and Figure 5B An aliphatic residue five or six amino acids amino-terminal to the P.Tyr is an important determinant for Shc PTB binding.
- GST-Shc PTB domain fusion proteins bound to glutathione-agarose were incubated with activated NGF receptors present in lysates of NGF-stimulated cells in the absence (lane 1) or presence (lanes 2-7) of 2 ⁇ M competing Wt and mutant phosphotyrosine containing peptides based on the sequence around Tyr 490, the Shc PTB domain binding site in the NGF receptor (lanes 2-5) or Tyr 960 an autophosphorylation site present within an Asn-Pro-X-P.Tyr motif in the insulin receptor (lanes 6 and 7).
- Wt NGF receptor peptide (Wt-NGFR, lane 2): H-I-I-E-N-P-Q-p.Y-F-S-D; Ala-6 NGF receptor peptide (NGFR-HAI): H-A-I-E-N-P-Q-p.Y-F-S-D; Ala-6, Ala-5 NGF receptor peptide (NGFR-HAA): H-A-A-E-N-P-Q-p.
- Y-F-S-D Ala-6, Ser-5 NGF receptor peptide (NGFR-HAS): H-A-S-E-N-P-Q-p.Y-F-S-D; Wt insulin receptor peptide (Wt-IR): Y-A-S-S-N-P-E-p.Y-L-S-A; Ile-5 insulin receptor peptide (IR-YAI): Y-A-I-S-N-P-E-p.Y-L-S-A. Bound proteins were analyzed by P.Tyr. blotting. Figure 5B.
- FIG. 6A Y-S-V-M-R-S-K).
- Figure 6B The requirement for an Arg residue at position 175 in the human Shc PTB domain has been conserved in evolution.
- Figure 6A GST fusion proteins containing Wt (lanes 1 and 2) or mutant (lanes 3-11) Shc PTB domains were incubated with NGF receptors present in lysates of control (lane 1) and NGF-stimulated cells (lanes 2-11). Bound proteins were analyzed by anti-P.Tyr blotting.
- Figure 6B The requirement for an Arg residue at position 175 in the human Shc PTB domain has been conserved in evolution.
- Figure 6A GST fusion proteins containing Wt (lanes 1 and 2) or mutant (lanes 3-11) Shc PTB domains were incubated with NGF receptors present in lysates of control (lane 1) and NGF-stimulated cells (lanes 2-11). Bound proteins were analyzed by anti-P.Tyr blotting.
- Figure 8 Peptides Competition in In Vitro Binding Assay. Cell Lysates were pre-incubated with 5 ⁇ M of appropriate peptides for 30 min. at 4°C. Then, proteins were precipitated by each binder, resolved on SDA-PAGE. Detection was carried out by anti-phospho-tyrosine antibody. Ins.:IRS-1 binding domain on insulin-R.
- Figure 9 Competition Assay of Penetrating Peptide.
- Cell lysates were pre-treated with appropriate peptides. Proteins were precipitated by GST-ShcB and detected by anti-phospho-tyrosine antibody. Peptides were prepared in DMSO solution.
- FIG. 10 Dose-Response Analysis of Peptides in in vitro Binding Assay.
- Cell Lysates were prepared and incubated with appropriate peptides in various concentrations. Proteins were precipitated by GST-ShcB and resolved on 10% SDS-PAGE gel. Anti-phospho-tyrosine antibody was used for detection.
- P-1 and P-2 peptides were dissolved in Hepes buffer (B), and in DMSO solution (C).
- FIG. 11 Proliferation of HER14 cells treated with peptides. Cells were starved for 24 (a) or 48 hrs (b) prior to stimulation. Cells were treated with appropriate peptide for 2 hrs, then stimulated with 100ng/ml EGF overnight. Cell proliferation was monitored by 3 H-TdR uptake.
- FIG. 12 Proliferation of HER14 cells.
- HER14 cells were cultured in serum-free D-MEM medium in 96-well plates for 24 hrs. Appropriate peptides were added in various concentrations 2 hrs prior to EGF stimulation. Cell proliferation was induced with 100ng/ml EGF overnight, then monitored by 3 H-TdR uptake.
- FIG. 13 Proliferation of HER14 cells treated with cyclic peptides.
- Cells were starved for 48 hrs in serum-free D-MEM medium. Appropriate peptide was added in various concentrations and cultured for 2 hrs prior to EGF stimulation. Cell proliferation was induced with 100ng/ml EGF overnight and monitored by 3 H-TdR uptake.
- FIG. 14 Proliferation of SupM2 cells treated with peptides.
- SupM2 cells were starved in serum-free RPMI 1640 medium for 24 hrs in the indicated cell number (a) or 2 ⁇ 10 4 /well (b). Cells were treated with penetrating peptide in various concentrations for 2 hrs prior to cell stimulation. Cell proliferation was induced with 20% FPS overnight and monitored by 3 H-TdR pulse.
- FIG. 15 MAPK Activation on PC12 Cells Treated with Peptides.
- PC12 cells were treated with appropriate peptide at various concentrations. Cells were stimulated with 50ng/ml NGF for 5 min., then cell lysates were prepared by standard methods. Each lane contains lO ⁇ g protein and MAPK (Erk-1) was detected by anti-Erk-1 polyclonal Ab. Arrows represent activated Erk-1/2 (b).
- Figure 16 Detection of Activated MAPK on PC12 Cells Treated with Peptides.
- PC 12 cells were treated with peptides for 4 hrs prior to stimulation. Cells were stimulated with 50ng/ml NGF for 5 min. and cell lysates were prepared according to standard methods.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU75211/96A AU7521196A (en) | 1995-10-27 | 1996-10-24 | Peptide inhibitors of a phosphotyrosine-binding domain containing protein |
EP96937741A EP0874639A4 (en) | 1995-10-27 | 1996-10-24 | Peptide inhibitors of a phosphotyrosine-binding domain containing protein |
JP9516793A JPH11514381A (en) | 1995-10-27 | 1996-10-24 | Peptide inhibitors of phosphotyrosine binding domain containing proteins |
US09/051,934 US6028053A (en) | 1995-10-27 | 1996-10-24 | Peptide inhibitors of a phosphotyrosine-binding domain containing protein |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US594495P | 1995-10-27 | 1995-10-27 | |
US1038496P | 1996-01-22 | 1996-01-22 | |
US1179996P | 1996-02-20 | 1996-02-20 | |
US60/010,384 | 1996-02-20 | ||
US60/005,944 | 1996-02-20 | ||
US60/011,799 | 1996-02-20 |
Publications (1)
Publication Number | Publication Date |
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WO1997015318A1 true WO1997015318A1 (en) | 1997-05-01 |
Family
ID=27357985
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/017080 WO1997015318A1 (en) | 1995-10-27 | 1996-10-24 | Peptide inhibitors of a phosphotyrosine-binding domain containing protein |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0874639A4 (en) |
JP (1) | JPH11514381A (en) |
AU (1) | AU7521196A (en) |
CA (1) | CA2235756A1 (en) |
WO (1) | WO1997015318A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998034954A2 (en) * | 1997-02-06 | 1998-08-13 | Mount Sinai Hospital Corporation | Ligands for discoidin domain receptor tyrosine kinases and complexes thereof |
WO1999027088A2 (en) * | 1997-11-19 | 1999-06-03 | Mount Sinai Hospital | Novel gene and protein expressed in neural and pancreatic tissues |
AU782304B2 (en) * | 1999-09-02 | 2005-07-14 | Acologix, Inc. | Methods and compositions for reducing serum phosphate levels |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US5352660A (en) * | 1991-10-31 | 1994-10-04 | Mount Sinai Hospital Corporation | Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system |
WO1995018823A2 (en) * | 1994-01-07 | 1995-07-13 | Beth Israel Hospital | Substrate specificity of protein kinases |
US5463023A (en) * | 1993-10-22 | 1995-10-31 | Washington University | Composition for inhibition of intracellular transcription |
WO1996011664A2 (en) * | 1994-10-10 | 1996-04-25 | Ludwig Institute For Cancer Research | Nsk2 a muscle receptor tyrosine kinase |
US5580979A (en) * | 1994-03-15 | 1996-12-03 | Trustees Of Tufts University | Phosphotyrosine peptidomimetics for inhibiting SH2 domain interactions |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6280964B1 (en) * | 1995-04-14 | 2001-08-28 | The Regents Of The University Of California | Binding sites for phosphotyrosine binding domains |
-
1996
- 1996-10-24 EP EP96937741A patent/EP0874639A4/en not_active Withdrawn
- 1996-10-24 CA CA002235756A patent/CA2235756A1/en not_active Abandoned
- 1996-10-24 AU AU75211/96A patent/AU7521196A/en not_active Abandoned
- 1996-10-24 WO PCT/US1996/017080 patent/WO1997015318A1/en not_active Application Discontinuation
- 1996-10-24 JP JP9516793A patent/JPH11514381A/en not_active Ceased
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5352660A (en) * | 1991-10-31 | 1994-10-04 | Mount Sinai Hospital Corporation | Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system |
US5463023A (en) * | 1993-10-22 | 1995-10-31 | Washington University | Composition for inhibition of intracellular transcription |
WO1995018823A2 (en) * | 1994-01-07 | 1995-07-13 | Beth Israel Hospital | Substrate specificity of protein kinases |
US5580979A (en) * | 1994-03-15 | 1996-12-03 | Trustees Of Tufts University | Phosphotyrosine peptidomimetics for inhibiting SH2 domain interactions |
WO1996011664A2 (en) * | 1994-10-10 | 1996-04-25 | Ludwig Institute For Cancer Research | Nsk2 a muscle receptor tyrosine kinase |
Non-Patent Citations (5)
Title |
---|
NATURE, 07 December 1995, Vol. 378, ZHOU et al., "Structure and Ligand Recognition of the Phosphotyrosine Binding Domain of Shc.", pages 584-592. * |
See also references of EP0874639A4 * |
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 04 August 1995, Vol. 270, No. 31, TRUB et al., "Specificity of the PTB Domain of Shc For betaTurnforming Pentapeptide Motifs Amino-Terminal to Phosphotyrosine", pages 18205-18208. * |
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 05 January 1996, Vol. 271, No. 1, LAMINET et al., "Affinity, Specificity and Kinetics of the Interaction of the SHC Phosphotyrosine Binding Domain with Asparagine-X-X-Phosphotyrosine Motifs of Growth Factor Receptors", pages 264-269. * |
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 05 November 1993, Vol. 268, No. 31, OBERMEIER et al., "Identification of Trk Binding Sites for SHC and Phosphatidylinositol 3'-Kinase and Formation of a Multimeric Signaling Complex", pages 22963-22966. * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998034954A2 (en) * | 1997-02-06 | 1998-08-13 | Mount Sinai Hospital Corporation | Ligands for discoidin domain receptor tyrosine kinases and complexes thereof |
WO1998034954A3 (en) * | 1997-02-06 | 1998-11-05 | Mount Sinai Hospital Corp | Ligands for discoidin domain receptor tyrosine kinases and complexes thereof |
WO1999027088A2 (en) * | 1997-11-19 | 1999-06-03 | Mount Sinai Hospital | Novel gene and protein expressed in neural and pancreatic tissues |
WO1999027088A3 (en) * | 1997-11-19 | 1999-08-12 | Mount Sinai Hospital Corp | Novel gene and protein expressed in neural and pancreatic tissues |
AU782304B2 (en) * | 1999-09-02 | 2005-07-14 | Acologix, Inc. | Methods and compositions for reducing serum phosphate levels |
Also Published As
Publication number | Publication date |
---|---|
EP0874639A4 (en) | 1999-07-28 |
CA2235756A1 (en) | 1997-05-01 |
AU7521196A (en) | 1997-05-15 |
EP0874639A1 (en) | 1998-11-04 |
JPH11514381A (en) | 1999-12-07 |
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