Note: Descriptions are shown in the official language in which they were submitted.
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PEPTIDE INHIBITORS OF A PHOSPHOTYROSINE-BINDING DOMAIN CONTAINING
PROTEIN
5 Fn~ n OF THE~ INVENTION
The invention relates to peptides which hl~elrele with the interaction of a
phn:,lJholylu.,ine-binding (PTB) domain C~ g protein with a PTB domain binding site;
and, uses of the peptides.
BACKGROUND OF THE INVENTION
Shc is a mlember 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 tr~n.~ductinn downstream of growth factor and
cytokine receptors (Pawson, 1995). Shc has been shown to bind to a wide variety of
15 activated growth iFactor and cytokine receptors. Shc was cloned from a human cDNA
library in a screen for SH2 domain-col 1~ g 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). O~ ssion of Shc results in cellular
2 o ~ rOlll lation of NIH3T3 fibroblasts and Ras-dependent neurite ~ulgl uwlll 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 phosphoLyl~s~l~e-binding (PTB) domain, a central
Pro-rich region tha~: contains the principal tyrosine phosphorylation site at Tyr 317, and an
25 SH2 domain at its carboxy-t~rminll~. The PTB domain, which is highly conserved in Shc-
related proteins, was recently identified based on its ability to bind to phosphotyrosine-
c~nt~ining proteins (Blaikie et al., 1994; Kavanaugh and Williams, 1994; van der Geer et
al., 1995). It recogni7~s pho~holyl~,si.le present within the sequence Asn-Pro-X-P.Tyr and
differs from SH2 domains that recognize phosphotyrosine in the context of carboxy-
3 o t~-rmin~l residues (l~avanaugh 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 stim~ tion with a wide variety
of growth factors and cytokines (Burns et al., 1993; Crowe et al., 1994; Cutler et al., 1993;
35 Lanfranconeetal., 1995;Peliccietal., 1992;Pronketal., 1993;Ravichandranetal., 1993;
Segatto et al., 1993; Yokote et al., 1994). Tyrosine phosphorylation of Shc is ~esP.nti 1l for
its interaction wil:h the Grb2-Sos complex, which may provide a me~h~ni~m for Ras
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activation (Buday and Duwllw~Ld, 1993; Crowe et al., 1994; Egan et al., 1993; Gale et al.,
1993, Li et al., 1993; Rozakis-Adcock e~ 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 l~C~l~. Several growth factomec~~ that had previously been shown to bind
5 to Shc upon activation contain tyrosine phosphorylation sites present within the sequence
Asn-Pro-X-P.Tyr, consistent with the notion that it is the PTB domain that mediates Shc's
interaction with these proteins (Campbell et al., 1994; van der Geer and Pawson, 1995).
Furthermore, 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
10 middle T antigen, and to a 145 kDa protein that becomes phosphorylated on tyrosine in
PDGF stim~ ted cells ~Blaikie et al., 1994; Kavanaugh and Williams, 1994; van der Geer
et al., 1995). The NGF lece~lor contains a single Shc-binding site at Tyr 490 that is present
within a Asn-Pro-X-Tyr motif (Obermeier et al., 1994; Stephens ef al., 1994). NGF
lt;c~l~ that have been m~lt~ted at Tyr 490 lack the ability to interact with Shc in vivo or
15 with the PTB domain in vilro (Stephens et al., 1994). Phosphotyrosine-cont~ining peptides
based on the Shc binding site in middle T antigen, which is also present within an Asn-Pro-
X-P.Tyr motif, compete with the NGF and EGF receptors for binding to the PTB domain
(van der Geer et al., 1995).
SUMMARY OF T~E INVENTION
2 0 The present inventors have idP.ntified the residues within the Asn-Pro-X-P.Tyr motif
of phospholyl~,sine-cont~ining proteins (e.g. growth activated growth factors and cytokine
receptors) that mediate the binding of the proteins to si~nS-lling proteins cont~inin~ PTB
domains. In particular, the present inventors found that the Asn and the phosphotyrosine
residues within the Asn-Pro-X-P.Tyr motif of the phosphotyrosine-cont~ining proteins
2 5 mediate their binding to the PTB domain of Shc. The present inventors also found that an
aliphatic residue that is five or six residues amino-terminal to the phosphotyrosine is
required for binding. This aliphatic residue is mi~in~ from the insulin receptorautophosphorylation site which is unable to form a stable complex with Shc. The present
illVt~ also analyzed the Shc PTB domain by in vitro mutagenesis and an evolutionarily~ o conserved Arg residue was identified that is important for PTB binding to its ligands.
Broadly stated the present invention relates to a peptide of the formula I
Xl - A' - A2- X2- Asn - X3- X4- P.Tyr - X5- X6- X7-XX
3 5 wherein Xl represents Lys, Arg, His, Ser, Thr, Tyr, Asn, Leu, Val, or Glu, Al represents
Trp, Leu, Ala, Ser, Ile, Glu, Met, Gly, Cys, Phe, Pro, or Val, and AZ represents Ala, Val,
Leu, Ile, Ser, Met, Phe, Gly, Cys, Trp, or Pro, X2represents Glu, Asn, Tyr, Thr, Ser, Asp,
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or Ile, X3 represents Pro, Met, Trp, Phe, Ala, Lys, Val, Leu, Ile, Gly, or Cys, X4 represents
Leu, Ala, Glu, Gln, Asp, Asn, Tyr, Thr, or Ser, X5 represents Phe, Trp, Pro, Leu, Ala, Val,
Ile, Gly, Cys, Met, Arg or Ser, x6 represents Ser, Thr, Tyr, Asn, Glu, Met, Ala, Leu, Val,
~ or Gly, X' represents Asp, Glu, Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro, Ser, or
5 Asn, and x8 which may be present or absent represents Phe, Trp, Pro, Leu, Ala, Val, Ile,
Gly, Cys, Met, Asp, Ser, or Arg, which interferes with the interaction of a PTB domain
cont~inin~ protein with a PTB domain binding site.
In an embodiment of the present invention a peptide of the formula I is provided:
Xl-Al-AZ-X2-Asn-X3-X4-P.Tyr-X5-X6-X7~Xg
wherein Xl represents His, Ser, Thr, Tyr, Asn, Leu, Val, or Glu, X2represents Glu, Ser,
Asp, or Ile, X3 represents Pro or Lys, X4 represents Leu, Ala, Glu, Gln, Asn, or Thr, Xs
represents Phe, Leu, Ile, Gly, Arg, or Ser, x6 represents Ser, Thr, Met, Ala, Leu, Val, or
15 Gly, X7 represents Asp, Ala, Val, Leu, Met, Ser, or Asn, x8 which may be present or
absent, represents ]Leu, Ala, Gly, Asp, Ser, or Arg, Al represents Trp, Leu, Ala, Ser, Ile,
Glu, Met, or Val, and A2 represents Ala, Val, Leu, Ile, Ser, Met, or Phe, which interferes
with the interaction of a PTB domain cont~ining protein with a PTB domain binding site.
In another embodiment of the invention a peptide of the formula Ia is provided
Xl - A' - A2- X2- Asn - X3- X4- P.Tyr - X5- X6- X7 Ia
wherein Xlrepresents Lys, Arg, His, preferably His, x2 represents Glu, Asn, Tyr, Thr, Ser,
preferably Glu, X3. represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, preferably
25 Pro, X4represents Gln, Asp, Asn, Tyr, Thr, Ser, preferably Gln, X5represents Phe, Trp,
Pro, Leu, Ala, Val, Ile, Gly, Cys, Met, preferably Phe, x6 represents Ser, Thr, Tyr, Asn,
Glu, preferably Ser, X' represents Asp, Glu, preferably Asp, and one of Al and A2
represents Ile and the other of Al and A2 represents Ile or Ala, preferably Al represents Ala
and A2 represents lle, which interferes with the interaction of a PTB domain cont~ining
3 o protein with a PTB domain binding site.
In still another embodiment of the invention a peptide of the formula Ia is provided
Xl - Al- A2- X2- Asn - X3- X4- P.Tyr - X5- X6- X7 Ia
wherein X' represents Ser, Thr, Tyr, Asn or Glu, preferably Tyr, x2 represents Glu, Asn,
35 Tyr, Thr, Ser, preferably Ser, X3represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys,
p~ ;l~ly Pro, X4 represents Glu, Asp, preferably Glu, X5 represents Phe, Trp, Pro, Leu,
Ala, Val, Ile, Gly, Cys, Met preferably Leu, x6 represents Ser, Thr, Tyr, Asn, Glu,
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preferably Ser, X' represents Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro, preferably
Ala, and one of Al and AZ represents Ile and the other of Al and AZ represents Ala, Val,
Leu, Ile, Gly, Cys, Phe, Trp, Met or Pro, which interferes with the interaction of a PTB
domain cont~ining protein with a PTB domain binding site.
The invention also relates to trlln~ti-~n~ 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 cont~ining protein with a PTB domain binding site;
and, ~h~rm~c~ltical col,lpositions for inhibiting the interaction of a PTB domain col-t~ ing
protein with a PTB domain binding site.
Further, the invention relates to a method of modlll~ting the interaction of a PTB
domain cQ~ g protein with a PTB domain binding site comprising rh~ngin~ the amino
acid Arg at position 175 in the PTB domain cont~ining protein. The invention still further
relates to a method for motl~ ting the interaction of an insulin receptor with insulin
receptor substrate 1 (IRS-l) or Shc comprising incorporating a large aliphatic amino acid
15 at amino acids -5 or -6 amino trrmin~l to the P.Tyr in the motif Asn-Pro-X-P.Tyr in the
PTB domain of the insulin receptor.
Other objects, features and advantages of the present invention will become apparent
from the following detailed description. It should be understood, however, that the detailed
description and the specific examples while indicating prefell~d embodiments of the
2 o invention are given by way of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent to those skilled in the art
from this detailed description.
CRIPTI~N OF THE DRAWINGS
The invention will be better understood with reference to the drawings in which:Figure lA is an immlln~blot showing P.Tyr-~.. ~ g proteins bound to GST (lane
2) and GST Shc PTB (lanes l, 3-6) fusion proteins immobilized on glutathione-agarose
after incubation with lysates from control (lane l) and NGF-stimul~ted (lanes 2-7) cells in
the absence (lane 1-3) and presence of Wt (lane 4) and mutant (lanes 5 and 6) competing
P.Tyr cont~inin~ peptides, based on the sequence around Tyr 490 the Shc PTB domain
3 o binding site in the NGF receptor;
Figure lB is the immlmnblot shown in Figure lA 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
35 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 srh~m~tic diagram showing the presence of an Asn-Pro-X-P.Tyr motif
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in the juxta membrane dom~in.~ of the NGF and insulin lece~L~
Figure 4A is an ;~ oblot showing anti-Shc immunopl~cipiL~les (lanes 1, 2, 5, 6,
9, and 10) from control (lanes 1, 5, and 9) and growth factor-stim~ ted (lanes 2, 6, and 10)
NIH3T3 fibroblasts ~ SSillg Wt (lanes 1 and 2; NGFR) or Phe 490 mutant (lanes 5 and
5 6; F490NGFR) NGF receptors, or CHO cells ~ ;ssi.lg Wt insulin receptors (lanes 9 and
10; IR) analyzed by anti-P.Tyr immlln~blotting; anti-NGF l~c~l-,r (lanes 3, 4, 7, and 8) and
anti-insulin lec~o~ "~ -precipit~tP~ (lanes 11 and 12) from control (lanes 3, 7, and 11)
and growth factor stiml-l~ted (lanes 4, 8, and 12) were analyzed in parallel;
Figure 4B is an i. . . ~ . .". .-)blot showing Wt (lanes 1 and 2) and Phe 490 mutant (5 and
10 6) NGF r~cel)lc,l~ present in lysates from control (lanes 1 and 5) and NGF-stim~ ted (lanes
2 and 6) cells e~ ~.7illg Wt (NGFR) or Phe 490 mutant (F490NGFR) and insulin receptors
(IR) present in Iysates from control (lane 9) and insulin-stiml-l~ted (lane 10) cells incubated
with GST-Shc PTB fusion proteins bound to gl~lt~thione-agarose~ bound proteins were
analyzed by anti-P.Tyr blotting;
Figure 5A is an immllnnblot showing GST-Shc PTB domain fusion proteins bound
to gll-t~t~ ne-agarose after incubation with activated NGF receptors present in lysates of
NGF-stiml-l~ted cells in the absence (lane 1) or presence (lanes 2-7) of 2 /lM competing Wt
and mutant phc-sphntyrosine co, I~ 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
2 0 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 thesequ~nce 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 960 in the insulin receptor (Y-A-S-S-N-P-E-p.Y-L-S-A; (O) and
2 5 substih~tion~ at position -5 and -6 with respect to the P.Tyr in the NGF receptor peptides (H-
A-S-E-N-P-Q-p.Y-F-S-D;(-)) and the insulin l~C~ul peptide (Y-A-I-S-N-P-E-p.Y-L-S-A;
(--) for their ability to compete for the binding of the GST-Shc PTB domain to the
immobilized polyoma middle T antigen peptide (L-S-L-L-S-N-P-T-p.Y-S-V-M-R-S-K);
Figure 6A is an immllnoblot showing GST fusion proteins cont~ining Wt (lanes 1
30 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 immllnl blot showing human EGF receptors bound to GST fusion
~ proteins cont~ining Wt (lanes 1 and 2) or Met 175 (lane 3) and Lys 175 (lane 4) mutant
3 5 human Shc PTB (lom~in~ in Iysates from control (lane 1) or EGF-stimlll~ted cells (lanes 2-
4) analyzed by anti-P.Tyr blotting, and in parallel GST (lane 8) and GST fusion proteins
cont~ining Wt (lane 7) or an Ala 151 mutant (lane 9) drosophila Shc PTB domain bound
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to ~hlt~thion~agarose~ in~ubat~d with fly lysates c~ ini~ 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 dom~in~ of m~mm~ n
5 and Drosophila Shc homologues;
Figure 8 are immlmoblots showing competitive inhibition of EGF receptor binding
to GST-ShcB analyzed by anti-phospho-tyrosine antibody;
Figure 9 is an immlmoblot showing co..~ e inhibition of EGF receptor binding
to GST-ShcB analyzed by anti-phospho-tyrosine antibody;
Figure 10 are immllnf~blots 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 HERl4 cells treated with cyclic
peptides of the invention;
Figure 14 are bar graphs showing proliferation of SupM2 cells treated with peptides
2 o of the invention;
Figure 15 are immunoblots showing MAPK activation on PC12 cells treated with
peptides of the invention; and
Figure 16 are immlmoblots showing activated MAPK on PC12 cells treated with
peptides of the invention.
25 D~ETAILEI) D~CRIPTION OF TEIE INVENTION
The following standard abbreviations for the atnino acid residues are used
throughout the specification: A, Ala - alanine; C, Cys - cysteine; D, Asp- aspartic acid; E,
Glu - ~hlt~mic 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,
3 0 Pro - proline; Q, Gln - glutamine; R, Arg - arginine; S, Ser - serine; T, Thr - threonine; V,
Val - valine; W, Trp- tryptophan; Y, Tyr - tyrosine; and p.Y., P.Tyr - phosphotyrosine.
As mentioned previously, the present invention relates to a peptide of the formula
I
Xl- Al - A2- X2- Asn - X3- X4 P.Tyr X5 X6 X7-xs
wherein Xl represents Lys, Arg, His, Ser, Thr, Tyr, Asn, Leu, Val, or Glu, Al represents
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Trp, Leu, Ala, Ser, Ile, Glu, Met, Gly, Cys, Phe, Pro, or Val, and AZ represents Ala, Val,
Leu, Ile, Ser, Met, Phe, Gly, Cys, Trp, or Pro, x2 represents Glu, Asn, Tyr, Thr, Ser, Asp,
or Ile, X3 represents Pro, Met, Trp, Phe, Ala, Lys, Val, Leu, Ile, Gly, or Cys, X4 represents
- Leu, Ala, Glu, Gln, Asp, Asn, Tyr, Thr, or Ser, X5 represents Phe, Trp, Pro, Leu, Ala, Val,
5 Ile, Gly, Cys, Met, Arg or Ser, x6 represents Ser, Thr, Tyr, Asn, Glu, Met, Ala, Leu, Val,
or Gly, X7 represents Asp, Glu, Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro, Ser, or
Asn, and Xg which may be present or absent represents Phe, Trp, Pro, Leu, Ala, Val, Ile,
Gly, Cys, Met, Asp, Ser, or Arg, which interferes with the interaction of a PTB domain
cont~ining protein with a PTB domain binding site.
lo In an embodiment of the present invention a peptide of the formula I is provided:
Xl-Al-A2-XZ-Asn-X3-X4-P.Tyr-X5-X6-X7~X8
wherein Xl represents His, Ser, Thr, Tyr, Asn, Leu, Val, or Glu, X2represents Glu, Ser,
15 Asp, or Ile, X3 represents Pro or Lys, X4 represents Leu, Ala, Glu, Gln, Asn, or Thr, X5
represents Phe, Leu, Ile, Gly, Arg, or Ser, x6 represents Ser, Thr, Met, Ala, Leu, Val, or
Gly, X7 represents Asp, Ala, Val, Leu, Met, Ser, or Asn, x8 which may be present or
absent, represents Leu, Ala, Gly, Asp, Ser, or Arg, Al represents Trp, Leu, Ala, Ser, Ile,
Glu, Met, or Val, and A2 represents Ala, Val, Leu, Ile, Ser, Met, or Phe, which interferes
2 0 .with the interaction of a PTB domain ct nt~inin~ protein with a PTB domain binding site.
In another embodiment of the invention a peptide of the formula Ia is provided
Xl-Al-A2-X2-Asn-X3-X4-P.Tyr-X5-X6-X' Ia
2 5 wherein Xl represents Lys, Arg, His, preferably His, x2 represents Glu, Asn, Tyr, Thr, Ser,
preferably Glu, X3 represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys, preferably
Pro, X4represents Gln, Asp, Asn, Tyr, Thr, Ser, preferably Gln, X5represents Phe, Trp,
Pro, Leu, Ala, Val, Ile, Gly, Cys, Met, preferably Phe, x6 represents Ser, Thr, Tyr, Asn,
Glu, preferably Ser, X7 represents Asp, Glu, preferably Asp, and one of Al and A2
3 o represents Ile and the other of Al and A2 represents Ile or Ala, preferably Al represents Ala
and A2 represents Ile, which interferes with the interaction of a PTB domain cont~ining
protein with a PTB domain binding site.
In still another embodiment of the invention a peptide of the formula Ia is provided
Xl-Al-A2-X2-Asn-X3_X4 p.Tyr-x5-x6-x7 Ia
wherein Xl represents Ser, Thr, Tyr, Asn or Glu, preferably Tyr, x2 represents Glu, Asn,
Tyr, Thr, Ser, preferably Ser, X3represents Pro, Met, Trp, Phe, Ala, Val, Leu, Ile, Gly, Cys,
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p~e,~bly Pro, X4 represents Glu, Asp, preferably Glu, Xs represents Phe, Trp, Pro, Leu,
Ala, Val, Ile, Gly, Cys, Met preferably Leu, x6 represents Ser, Thr, Tyr, Asn, Glu,
preferably Ser, X' represents Ala, Val, Leu, Ile, Gly, Cys, Phe, Trp, Met, Pro, preferably
Ala, and one of A' and AZ represents Ile and the other of A' and A2 represents Ala, Val,
5 Leu, Ile, Gly, Cys, Phe, Trp, Met or Pro, which hllt;;~r~s with the interaction of a PTB
domain cont~inin~? protein with a PTB domain binding site.
Preferred peptides of the illV~llLiOI- 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;
10 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-P.Tyr-Gly-Thr-Ser-Asp; Leu-Leu-Leu-Ser-Asn-Pro-Ala-P.Tyr.-Arg-Leu-Leu-Leu; Tyr-Ala-Ser-Ser-Asn-Pro-Glu-P.Tyr-Leu-Ser-
Ala-Ser; Val-Ser-Val-Asp-Asn-Pro-Glu-P.Tyr-Leu-Leu-Asn-Ala; Ser-Leu-Leu-Ser-Asn-Pro-Thr-P.Tyr-Ser-Val-Met-Arg; Asn-Glu-Met-Ile-Asn-Pro-Asn-P.Tyr-Ile-Gly-Met-Gly;
15 and Glu-Met-Phe-Glu-Asn-Pro-Leu-P.Tyr-Gly-Ser-Val-Ser (SEQ. ID. NOS. 1 -13 in the
Sequence Listing).
In ~ ition to full-length peptides of the formula I, truncations of the peptides which
inhibit interaction of PTB domain cont~inin~ proteins with PTB domain binding sites are
lated in the present invention. Truncated peptides may comprise peptides of about
2 o 7 to 10 amino acid residues. In an embodiment of the invention the truncated peptide has
the sequence A2-X2-Asn-X3-X4-P.Tyr or A2-X2-Asn-X3-X4-P.Tyr-X5 wherein A2, X2, X3, X4,
and X5 are as defined above. In a preferred embodiment of the invention, the truncated
peptide has the sequence Leu/Ile-X2-Asn-Pro-X4-P.Tyr, wherein x2 represents Glu, Ser,
Asp, or Ile, and X4 represents Leu, Ala, Glu, Gln, Asn, or Thr. Examples of truncated
2 5 peptides include Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe; Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser-Pro-
Gly; Ala-Glu-Asn-Pro-Gln-P.Tyr-Phe; Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Ser; Ile-Ser-Asn-
Pro-Glu-P.Tyr-Leu; Val-Leu-Ala-Asp-Asn-Pro-Ala-P.Tyr-Arg-Ser-Ala (SEQ. ID. NOs. 14
to 19 in the Sequence Listing).
The truncated peptides may have an amino group (-NH2), a hydrophobic group (for
3 0 example, carbobenzoxyl, dansyl, or T-butylo~yc~bonyl), an acetyl group, a 9-fluol~llyhllethoxy-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
3 5 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
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g
of the formula I cont~inin~ 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. ill~ r~;lt;llce with the interaction of a PTB domain co.l~ g protein with a PTB domain
binding site, and may further possess additional advantageous features such as increased
5 bioavailability, stability, or reduced host immune recognition.
One or more amino acid insertions may be introduced into a peptide of the formula
I preferably outside the sequence A2-X2-Asn-X3-X4-P.Tyr-X5. For example, amino acid
insertions may be made between Xl and Al or between X5 and X6, or X6 and X'. Amino
acid insertions may consist of a single amino acid residue or sequential amino acids.
0 One or more amino acids, preferably one to five 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-Asp-Asn-Pro-Glu-
P.Tyr-Leu-Leu-Asn-Ala-Gln-Lys; Leu-Ser-Leu-Leu-Ser-Asn-Pro-Thr-P.Tyr-Ser-Val-Met-
5 Arg-Ser-Lys; Val-Ser-Ser-Leu-Asn-Glu-Met-Ile-Asn-Pro-Asn-P.Tyr-Ile-Gly-Met-Gly-Pro-
Phe; and Leu-Leu-Leu-Thr-Lys-Pro-Glu-Met-Phe-Glu-Asn-Pro-Leu-P.Tyr-Gly-Ser-Val-
Ser-Ser-Phe (SEQ. ID. NOs. 20 to 25 in the Sequence Listing).
Deletions may consist of the removal of one or more amino acids, or discrete
portions from the peptide sequPnce pl~rel~ly outside the A2-X2-Asn-X3-X4-P.Tyr sequence.
2 0 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.
It is anticipated that if amino acids are inserted or deleted in sequences outside the
A2-X2-Asn-X3-X4-P.Tyr sequence that the resulting analog of the peptide will exhibit the
activity of a peptidle of the invention.
Cyclic delivaLives of the peptides of the invention are also part of the presentinvention. Cyclization may allow the peptide to assume a more favorable confollllation for
association with a PTB domain cont~ining protein. Cyclization may be achieved using
techniques known in the art. For example, disulf1de bonds may be formed between two
appl~,pliately spaced components having free sulfhydryl groups, or an amide bond may be
30 formed between an amino group of one colllponent and a carboxyl group of another
component. Cyclization may also be achieved using an azobenzene-cont~ining 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
3 5 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-
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-- 10 --
Gly) (SEQ. ID. NOs. 26 to 27 in the SçquP.nce Listing) where the amino group of isoleucine
and the carboxyl group of glycine forrn a peptide bond; cyclo-(His-Ile-Ile-Glu-Asn-Pro-
Gln-P.Tyr-Phe-Ser-Asp-Ala-Pro-Gly) (SEQ. ID. NO. 28 in the Sequence Listing) where
the amino group of histidine and the carboxyl group of glycine form a peptide bond; and
5 Cys-Ile-Ile-Glu-Asn-Pro-Gln-P.Tyr-Phe-Cys (SEQ. ID. NO. 29 in the Sequence Listing)
having a disulphide bond between the two cysteine residues.
In an embodiment of the invention, 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
0 cyclic peptide is a peptide of the invention with an Ile in the left position (i.e. a terminal Al
or A2is Ile) and the amino acids Pro-Gly at the right position. The amino group of the Ile
and the c~hl u~yl group of the Gly forrn a peptide bond r~ lltinp in a cyclic peptide. The 3D
structure of the cyclic peptide is similar to the original structure of the PTB binding site of
TrkA.
The following is an example of a 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). In ~is peptide, Asn-Pro-Gln-P.Tyr take a native beta-turn, Ser-Pro-Gly-
Ile make another beta-turn on the other side, and the central part adopts an allLip~ ~llel beta-
sheet. A beta-sheet has two faces, and the peptide binds to the PTB domain with the face
2 o 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.
It may be desirable to produce a cyclic peptide which is more flexible than the cyclic
peptides co"~ ;,.g.peptide bond link~g~ as described above. A more flexible peptide may
2 5 be pLepaL ed by introducing cysteines at the right and left position of the peptide and forming
a ~ lphide 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
di.~ulfitle linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The
relative flexibility of a cyclic peptide can be det~rmin~d by molecular dynamics ~imlll~tions.
3 o The invention also includes a peptide conjugated with a selected peptide, protein,
or a selectable marker (see below) to produce fusion proteins. For exarnple, a peptide of the
invention may be conjugated with a peptide which facilitates entry into cells.
The peptides of the invention may be ~)llV~l L~d into ph~ celltical salts by reacting
with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric
35 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.
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The peptides of the invention may be prepared using recombinant DNA methods.
Accordingly, nucleic acid molecules which encode a peptide of the invention may be
incol~ol~L~d in a kno~,vn manner into an appl.,pliate cA~l-ession vector which ensures good
expression of the peptide. Possible l;;xpl~ssion vectors include but are not limited to
5 co~mitl.c, pl~mi(1s, 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 n.ece~u,y regulatory sequPncPs for the transcription and translation of the
inserted protein-sequence. Suitable regulatory sequences may be obtained from a variety
of sources, in~ in5~ bacterial, fungal, viral, m~mm~ n, or insect genes (For example, see
10 the re~ tc)~ sequences described in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, ~c~(1Pnnic Press, San Diego, CA (1990). Selection of app~opliateregulatory 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, ~nh~nc~rs, and sequences conferring inducibility of
15 transcription may also be incol~ol~led into the ~AI~ression vector.
The recombinant eA~ ssion vectors may also contain a selectable marker
gene which f~cil;t~tP,q 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,
20 firefly luciferase, or an immllnnglobulin 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 ~A~ ssion of the recombinant peptide; increased25 solubility of the recombinant peptide; and/or aid in the purif1cation of the recombinant
peptide by acting as a ligand in affmity purification. For example, a proteolytic cleavage
site may be inserted in the recombinant peptide to allow separation of the recombinant
peptide from the fusion portion after purif1cation of the fusion protein. Examples of fusion
expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New
30 Fn,~l~n~1 Biolabs, Beverly, MA) and pRlT5 (Ph~rrn~ , PiscaL~w~y, NJ) which fuse
glutathione S-tran.sferase (GST), maltose E binding protein, or protein A, respectively, to
- the recombinant protein.
Recombinant t;A~.lession vectors may be introduced into host cells to produce a
- transformant host cell. Transformant host cells include prokaryotic and eukaryotic cells
3 5 which have been transformed or kansfected with a recombinant expression vector of the
invention. The terms "transformed with", "transfected with", "transformation" and
"transfection" are int~n~lecl to include the introduction of nucleic acid (e.g. a vector) into a
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-- 12
cell by one of many techniques known in the art. For example, prokaryotic cells can be
transformed with nucleic acid by electroporation or calcium-chloride mediated
transformation. Nucleic acid can be introduced into m~mm~ n cells using collvenlional
techniques such as calcium ph~.~ph~t~ or r~lcillm chloride co-precipitation, DEAE-dextran-
5 mediated transfection, lipofectin, ele~ upol~lion or microinjection. Suitable methods fortransforming 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.
10 For example, the peptides of the invention may be expressed in bacterial cells such as E.
coli, insect cells (using baculovirus), yeast cells or m~mm~ n cells. Other suitable host
cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185,
~Ac~ .mic Press, San Diego, CA (1991).
The peptides of the invention may be tyrosine phosphorylated using the method
15 described in Reedijk et al. (The EMBO Journal 11 (4): 1365, 1992). For example, tyrosine
phosphorylation may be induc.e~ by infecting bacteria harbouring a plasmid cont~ining 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
cont~ining the plasmid and bacteriophage as a lysogen are isolated. Following induction
2 0 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(Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous
solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I
25 and II, Thieme, Stuttgart). By way of example, the peptides may be synthesi7ecl using 9-
fluorenyl methvxyc~bonyl (Fmoc) solid phase chemistry with direct incorporation of
phosphotyrosine as the N-fluolenyllllethoxy-carbonyl-O-dimethyl phosphono-L-tyrosine
derivative.
N-terminal or C-terminal fusion proteins comprising a peptide of the invention
3 o 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 rçslllt~nt fusion
proteins contain the peptide fused to the selected protein or marker protein as described
herein. Examples of proteins which may be used to prepare fusion proteins include
35 immlln~,globulins, glutathione-S-transferase (GST), h~m~h~ (HA), and truncated
myc.
The peptides of the invention may be used to prepare monoclonal or polyclonal
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-- 13
antibodies. Convel~tional methods can be used to prepare the antibodies. As to the details
relating to the L~ Lion of monoclonal antibodies reference can be made to Goding, J.W.,
Monoclonal Antibodies: Principles and Practice, 2nd Ed., Ac~ mic Press, London, 1986.
As ~ cussed below, the antibodies may be used to identify proteins with PTB domain
5 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 m~teri~, and r~-lio~ctive material are well known to the skilled artisan.
10 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 cont~inin~ proteins such as Shc.
The peptides of the invention interfere with the interaction of a PTB domain
cont~inin~ protein and a PTB domain binding site. The term "PTB domain cont~inin~
15 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.
270:15639-15643; van der Geer, P., & Pawson, T., 1995, TIBS 20:277-280; Batzer, A.G.,
et al., Mol. Cell. Biol. 1995, 15:4403-4409; and Trub, T., et al., 1995, J. Biol. Chem.
270: 18205-18208; van der Geer et al., Current Biology 5(4):404, 1995)). 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.
Examples of PTB domain cont~ining proteins are m~mm~ n 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 cont~inin~; proteins
may also be identified by screening a cDNA c~p~ ,ion library with a protein cont~inin~ a
30 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 con~ining proteins may also
be screened using antibodies specific for the PTB domain. For example, 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 ~ ession library with proteins based on the consensus
35 seq~l~nt~e 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, Januar,v 1987) with the PTB domain specific probe can be used.
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The term " PTB domain binding site" refers to a sequence with high affinity to PTB
dc m~in~. 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
5 Williams, 1994; van der Geer ef al., 1995; Damen et al., 1996), ErbB2, ErbB3, TrkA, TrkB,
TrkC, MCKlOb, insulin receptor, IGF-l receptor, and IL-4 recepton PTB domain binding
sites may be identified by screening with PTB domain cont~inin~ proteins or withantibodies specific for the peptides of the invention.
The phrase "hl~lrele with the interaction of" refers to the ability of the peptides of
10 the invention to inhibit the binding of a PTB domain cont~ining protein to a PTB domain
binding site thereby affecting regulatory palllw~y~ that control gene ~ lession, cell
division, cytoskeletal architect~lre and cell metabolism. Examples of such regulatory
pathways are the Ras pathway, the pathway that regll~t~ the breakdown of
polyphosphninositides through phospholipase C, and PI-3-kinase activated pathways, such
15 as the rapamycin-sensitive protein kinase B (PKB/Akt) p~ w~y~
The peptides of the invention have been specifically shown to interfere with theinteraction of the PTB domain of Shc and ph.~ hl .lyl~shle-~nt~ining 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 ~nti~n Accordingly, the activity of a peptide of the
2 O invention may be confirmed by assaying for the ability of the peptide to interfere with the
interaction of the PTB domain of Shc and I~h~)~photyrosine-cnnt~ining 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
25 interaction of a peptide of the invention, and truncations and analogs thereof with a PTB
domain ~."J~;..;..~ 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 phospholylusille-cont~ining
3 o peptides as discussed above.
The peptides of the invention mediate the interactions of PTB domain cont~inin~
proteins with PTB domain binding sites on proteins such as growth factors and cytokine
~:cel-L,l~ which regulate p~Lllw~ys that conkol gene ~L,lession, cell division, cytoskeletal
architecture and cell metabolism. The peptides may therefore be used in the keatment of
3 5 conditions involving perturbation of such regulatory palllw~y~. In particular, the peptides
may be useful in treating disorders involving excessive proliferation inc.l~ in~ various forms
of cancer such as leukemias, lymphomas (Hodgkins and non-Hodgkins), sarcomas,
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-- 15
melanomas, aderlomas, 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,
5 arthrosclerosis, angiogenesis, and viral infections, in particular HIV infections; and
- ~uLul.. ~ e diseases including systemic lupus erythematosus, Wegener's granulom~tosie7
rheumatoid arthritis, sarcoidosis, poly~ ~ is, pemphigus, pemphigoid, erythema
multiforme, Sjogren's syndrome, infl~mm~tory bowel disease, multiple sclerosis,
my~eth~ni~ gravis, keratitis, scleritis, Type I diabetes, insulin-dependent diabetes mellitus,
10 Lupus Nephritis, and allergic encephalomyelitis.
The invention also relates to a ph~rm~e~ltical composition comprising a peptide of
the invention foruse as an ~"1;.~ ni.~l of the interaction of a PTB domain cont~ining protein,
preferably Shc and a PTB domain binding site, preferably an activated growth factor or
cytokine receptor.
15The peptides of the invention may be form~ ted into pharm~ce ltical compositions
for adminstration to subjects in a therapeutically active amount and in a biologically
compatible form suitable for ~l1mini~etration in vivo i.e. a form of the peptides to be
?~rlmini.etered in which any toxic effects are outweighed by the thel~uLic effects.
The peptides may be a-lmini.etered to living org~niem.e including hl-m~ne7 and
20 ~nim~le. A thera]peutically active amount of the ph~rm~ce~ltical compositions of the
invention is defined as an amount effective, at dosages and for periods of time necessary to
achieve the desired result. For example, 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.
25The peptides may be ~imini~etered in a convenient manner such as by injection
(subcutaneous, intravenous, etc.), oral ~mini.etration, inhalation, transdermal application,
or rectal ~timinietration. Depending on the route of ~lmini~etration7 the peptides may be
coated in a m~t.o.ri~l to protect them from the action of enzymes. The peptides may also be
used in combination with organic substances for prolongation of their ph~rm~cologic
3 o actions. Examples of such organic substances are non-antigenic gelatin,
c~b~xy..lethylcellulose, sulfonate or phosphate ester of alginic acid, dextran, polyethylene
glycol and other glycols, phytic acid, poly~,lul~nic acid, and protamine.
The compositions described herein can be prepared by ~L~ known methods for
- the p-~ Lion of ph~rm~e~ltically acceptable compositions which can be ~(lmini.etered to
35 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
Remin ton~s Ph~rm~ce~ltical Sciences (pcemingttln~s Ph~rm~ce~ltical Sciences, Mack
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-- 16
Publishing Company, Easton, Pa., USA 1985). On this basis, the cc,~ )osilions include,
albeit not exclusively, solutions of the peptides in association with one or more
ph~ tir.~lly 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
5 incoll ol~L~d in liposomes or similar delivery vehicles.
The utility of the peptides and compositions of the invention may be conrll ' lled in
in vitro cell penetration assays. For example, the effects of the peptides upon cellular
filnction~ in vivo may be confirmed using electroporation techniques (See Raptis, L., and
K.L. Firth, DNA and Cell Biology, 9:615, 1990 and Raptis, L.H. Et al., BioTechniques
10 18:104, 1995).
The utility of the peptides and compositions of the invention may also be confirmed
in in vivo animal experimental model systems. For example, therapeutic utility in
proliferative disorders may be tested by ~xs..~ lg the ability of a substance to suppress the
growth of a transplantable tumor. Particular in vivo animal models which may be used
15 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
M M T V-neu transgenes, which result in the development of m~mm~ry carcinoma.
The following non-limiting examples are illustrative of the present invention:
h,xAMPLl~S
2 0 E~cample 1
The following materials and methods were utilized in the investigations outlined in
the example:
MATERIALS AND MET13:ODS
Cell lines, anti-sera and fusion proteins. CHO cells ~ ssiag Wt insulin receptors
25 (White et al., 1988) were grown in F12 medium cont~inin g 25 mM Hepes pH 7.4, and 10%
fetal bovine serum. NIH3T3 cells ~x~J.essillg Wt and Phe 490 mutant NGF receptor(Stephens et al., 1994) were grown Dulbecco-Vogt's modified Eagle medium (DMEM)
cont~ining 10% calf serum (CS). NIH3T3 cells ovel~x~ ssillg the human EGF receptor
(Honegger et al., 1987) were grown in DMEM cont~inin~ 10% CS and 400,ug/ml G418.30 The monoclonal anti-insulin l~ce~ r antibody 51 was obtained from Dr. I. Goldfine
~Fol~y~lllet al., 1987; Roth et al., 1982). A polyclonal anti-NGF receptor antiserum was
raised against NGF receptor carboxy-t~rmin~ls (Hempstead et al., 1992), the anti-Shc
polyclonal serum was raised against a GST-Shc SH2 domain fusion protein. The anti-P.Tyr
monoclonal Antibody 4G10 was obtained from UBI (Lake Placid NY). The GST-Shc PTB3 5 fusion protein used in the lecep~Jr binding experiments described here is identical to GST-
ShcB described in van der Geer et al., 1995. The GST-dShc PTB fusion protein has been
described previously (Lai et al., 1995).
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Immunopr~c;l,ilations and PTB binding assays. Cells were grown to confluence andstarved 16 hr in medium without serum. CHO cells e?~ ssillg the insulin le;cep~ur were
sfim~ ted with 100 nM insulin for 5 min at 37~C. NIH3T3 cells c~ ing NGF receptors
were stim~ ted with 50 ng/ml NGF for 5 min at 37~C, and NIH3T3 cells expressing the
5 human EGF l~c~Lur were stim~ te~l with 100 ng/ml EGF for 5 min at 37~C. Control and
growth factor stim~ ted 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 MgCl2, 1 mM
EGTA, 100 mM NaF, 10 mM Sodium Pyrophosphate, 500 ,uM Sodium V~n~te, 1 mM
PMSF, 10 ,ug/ml Aprotinin, and 10 ,ug/ml Leupeptin (PLC-lysis buffer) per 10 cm dish.
:Lo Immunoprecipitations and PTB-binding assays in the absence or presence of 2 or 5 ~I
~lllpe~ g phosphopeptide were performed exactly as described previously (van der Geer
etal., 1995).
Surface Plasmon R~.~or ~nce analysis of phosphopeptides interacting with the Shc PTB
domain. Peptides were synth~i7ed using 9-Iluol~llyl methoxycarbonyl (Fmoc) solid phase
15 chemistry with direct incorporation of phosphotyrosine as the N~-fluorenylmethoxy-
carbonyl-O-dimethyl-phosphono-L-tyrosine derivative. Cleavage of the peptide from the
resin and d~lule~;lion was achieved through an 8 hr incubation at 4~C in trifluoroacetic acid
cont~inin~ 2 M bromotrimethyl silane and a scavenger mixture composed of thioanisole,
m-cresol and 1,2-eth~n~lithit l (1 .û: 0.5: 0.1% by volume). The product was precipil~L~d
2 0 with cold t-butyl ethylether and collected by centrifugation. Following desalting of the
crude material, pure phosphopeptide was isolated using reverse phase HPLC. The
~llthenticity of the phosphopeptide was confirmed by amino acid analysis and mass
spectroscopy.
Surface pllasmon resonance analysis was carried out using a Biacore apparatus
2 5 (Ph~rrn~ri~ Biosensor) as described previously (Puil et al., 1994). The peptide L-S-L-L-S-
N-P-T-p.Y-S-V-M-R-S-K was immobilized to a biosensor chip through injection of a 0.5
mM solution of the phosphopeptide, in 50 mM HEPES, pH 7.5 and 2 M NaCl, across the
chip surface previously activated following procedures outlined by the manufacturer.
Inj ection of anti-phosphotyrosine antibody was used to confirm that successful
30 immobilizationofthepeptidewas achieved. Solutions (100 ,ul) cont~ining 1 ,uM GST-Shc
PTB domain fusion protein and the indicated concentrations of soluble phosphopeptide in
~ 50 mM Na phosphate7 pH 7.5, 150 mM NaCl, 0.1 mM EDTA, and 2 mM DTT, were
injected across the surface. The amount of bound GST-Shc PTB domain was estimated
- from the surface plasmon r~ n~nce signal at a fixed time following the end of the injection
3 5 and the percentage bound, relative to injection of GST-Shc PTB domain alone, calculated.
The surface was regenerated using 2 M Guanidinium-HCl.
Expression of Torso-DER in transgenic flies. Transgenic flies ~ esshlg the activated
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-- 18 --
Torso-DER chimeric protein expressed under the control of the heat shock promoter were
obtained and protein cAI)lession was intlllced 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 ef al., 1995).
5 I. Ide.llirying Motifs RecG~ ed by the Shc PTB Domain
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 reco ni7:ed by the PTB domain, it was shown that
peptides that contain a phn:~lJhnlyl~sine within the sequence Asn-Pro-X-P.Tyr can compete
0 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 et al., 1995). To investigate the contribution of the
Asn and Pro residues within the consensus PTB domain binding site to phosphopeptide
recognition, the residues were changed to Ala in a phosphopeptide based on the sequence
15 around Tyr 490, the Shc-binding site in the NGF receptor. Wt and mutant peptides were
tested for their ability to ~1~ ~ with NGF lec~Lul~, present in lysates of NGF-stim~ ted
cells, for binding to a GST fusion protein cnnt~ining the Shc PTB domain (Figure lA).
Bound proteins were detectec~ by anti-P.Tyr immlm~blotting. Only the activated NGF
receptor bound the Shc PTB domain in vitro. The Wt phosphopeptide (His-Ile-Ile-Glu-Asn-
20 Pro-Gln-P.Tyr-Phe-Ser-Asp) competed efficiently for binding. Ch~n.~in~ the Asn at
position -3 (relative to the P.Tyr) to Ala completely abolished binding, whereas çh~nging
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 polyclonalantiserum raised against the NGF lece,o~ul (Figure lB). To confirm these results and to
25 estimate the contribution of the different residues more precisely, a wide range of
conc~.ntrations of the different peptides was tested for their ability to inhibit binding of the
Shc PTB domain to a phosphotyrosine-cont~ining peptide, based on the sequence around
Tyr 250 the Shc-binding site in polyoma middle T antigen, immobilized on a Biacore chip
(Figure 2). The data show that within the Asn-Pro-X-P.Tyr motif the Asn residue is
3 o essential for peptide binding by the PTB domain; presence of the Pro residue further
increases the affinity applo~hllately ten fold (Table 1). PTB binding depends onphn~l~hn~ylation of the Tyr residue present within the consensus binding site (Blaikie et al.,
1994; Kavanaugh and Williams, 1994; van der Geer et al., 1995). These results are
cc n~i~t.o.nt with the presence of Asn-Pro-X-P.Tyr motifs in a variety of receptors for growth
3 5 factors and cytokines that have been shown to bind Shc.
:C[. Why the Insulin Receptor Lacks the Ability to bind to Shc
The insulin receptor, which cont~in~ a bona fide autophosphorylation site that is
CA 0223=,7=,6 1998-04-24
W ~ 97/15318 PCT~US96/I708a
-- 19 --
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
5 associate stably with Shc was confirmed in coimmllnc-precipitation experiments in which
~ Shc immllnoprecipiL~Les were analyzed for associated proteins by anti-P.Tyr
immllnnblotting (Figure 4A). Wt but not Phe 490 mutant NGF receptors can be ~letected
in Shc immlmnp~ iL~Les from NGF-stimlll~ted cells. In contrast, insulin receptors were
absent from Shc i~ c;cipitates from insulin stimlll~ted CHO cells overexpressing the
10 Wt insulin l~ceplor (CHO-IR cells). To test whether the insulin receptors inability to
associate with Shc in cells was reflected in an inability to bind to the Shc PTB domain in
vitro, GST fusion proteins cont~inin~: the Shc PTB domain were incubated with Iysates of
control and insulin-stiml-l~ted CHO-IR cells and bound proteins were vi.~u~li7ed by anti-
P.Tyr immlln- blotling. Wt and Phe 490 NGF l~c~L~ , were inclllded as controls. The NGF
15 lt;c~Lor bound to the Shc PTB domain in vi~ro (Figure 4B) and binding was dependent on
phosphorylation of the NGF 1 ec~L~r at Tyr 490. In contrast, no tyrosine phosphorylated
insulin receptors were bound to the Shc PTB domain in vitro (Fig,ure 4B).
To investigate the possibility that access to the Asn-Pro-X-P.Tyr motif in the insulin
receptor is blocked, the ability of the phosphotyrosine-cont~ining peptide based on the
20 sequence around Tyr 960 in the insulin receptor to compete with the NGF receptor for
binding to the Shc PTB domain was tested. In co"LI~L to the NGF receptor
phn.crhopeptide, the insulin lt;c~L~l peptide was unable to compete (Fig,ure 5A, lanes 2 and
6, and Figure SB). This indicates that the inability of the insulin receptor to bind the PTB
domain is retained in this phosphopeptide that starts seven amino acid residues amino-
25 terminal to the P.Tyr (Table 1). The NGF lt;ceptor and several other proteins with welldefined Shc-binding sites often contain large aliphatic residues at six and five residues
amino-t~rrnin~l of the rh~,sph~lrylated 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
3 o binding, several substitntion~ 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. Ch~nging the Ile six
residues u~sLlealll of the P.Tyr to an Ala had no effect on the ability to bind to the PTB
- domain (Figure 5A and 5B). In collLl~L, çh~n~ing the Ile at -5 to Ala in addition to
3 5 ch~nging the Ile at -6 reduced the ability to bind to the PTB domain (Figure 5A and 5B).
(~h~nging the Ile residues at -5 to a Ser in addition to çh~ngin~ the Ile at -6 to Ala, identical
to what is found in the insulin receptor, abolished binding (Figure 5A and 5B). These data
CA 0223~7~6 1998-04-24
WO 97/15318 PCT/US96/17080
-- 20 --
clearly implicate the aliphatic residues five and six residues amino-t~rmin~l to the
phosphotyrosine in the PTB-binding site as being important for binding to the Shc PTB
domain and suggest that ~~h~n~ing the Ser, five residues u~ e~ll of the P.Tyr in the insulin
receptor peptide, to an Ile should increase its ability to bind to the PTB domain
5 dramatically. This was tested and it was found that in contrast to the Wt insulin receptor
peptide, which has no measurable affinity for the PTB domain, the mutant insulin receptor
peptide competed efficiently with the NGF receptor and the NGF receptor peptide for
binding to the PTB domain (Figs. 5A and 5B). The data presented here (sllmm~rized in
Table 1) indicate that the Shc PTB domain specifically recognizes P.Tyr residues in the
10 context of a Asn at -3 and a large aliphatic at -5 or -6. A P,ro residue at -2 increases the
affinity but appears to be non-essential.
m. Characterization of the PTB Domain of Shc
A comparison of the PTB dom~in~ present in Shc and its relatives revealed the
presence of a large number of conserved Arg residues. Several conserved Arg are directly
5 involved in P.Tyr binding by SH2 domains. As an initial attempt to characterize PTB
domain P.Tyr-binding, all conserved Arg residues in the Shc PTB domain were individually
mutated and GST-fusion proteins co~ i"i~lg mutant PTB domains were tested for their
ability to bind to the activated NGF l ec~r (Fig. 6A). The three Arg residues and the Lys
that are present between residues 97 and 100 were mut~ted to Met in combination. Of all
2 o ten Arg residues tested, only mutation of Arg 175 had a dramatic effect on the affmity of
the Shc PTB domain for the activated NGF receptor (Figure 6A). Both the Met 175 and the
Lys 175 ~.~uL~ were strongly impaired in their binding activity (Figures 6A and 6B),
indicating that not just a positive charge but a positive charge in the context of an Arg
residue is required at this position. The dShc PTB domain contains an Arg at residue 151,
25 which is homologous to Arg 175 in the human Shc. Wt and mutant dShc PTB domains
were tested for their ability to bind to the drosophila EGF receptor (DER) (Figure 6B). The
ability of Wt and the 175 mutant human Shc PTB domains to bind to the human EGF
~ ;c~c,r were tested in parallel (Figure 6B). The Wt dShc PTB domain but not the Arg to
Ala mutant at position 151 was able to bind efficiently to activated DER in vitro, suggesting
3 0 that the requirement for the presence of an Arg residue at position 175 in the human Shc
PTB domain has been conserved in evolution.
Summary
Shc binding to activated growth factor receptors appears to be an important step in
the initiation of signal tr~ncd~lction towards DNA synthesis and cell division or
3 5 differentiation. Shc binding sites are particularly well characterized in the NGF receptor
and in polyoma middle T ~ tigen. In 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 PLCy-
CA 0223~7~6 1998-04-24
WO 97/15318 PCI~/US96/17080
-- 21
binding site, completely blocks NGF-in~ ced neuronal di~rele~llLiation in PC12 cells
(Stephens ef al., 1994). Mutation of Tyr 250, which is the Shc binding site, in polyoma
middle T antigen blocks cellular ~ llation (Campbell et al., 1994; Dilworth et al.,
~ 1994). The EGF receptor also interacts strongly with Shc, although the precise contribution
5 of different autophosphorylation sites in the EGF receptor carboxy-t~ nll~ remains
~ unresolved (Batzer et al., 1994; Okabayashi et al., 1994).
The PTB domain at the amino-tPrmim-.~ 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-cont~inin~ peptides compete
0 efficiently for Shc PTB binding to activated growth factor receptors, such as the receptors
for EGF and NGF (Blaikie ef al., 1994; Campbell et al., 1994; Kavanaugh et al., 1995; van
der Geer and Pawson, 1995; van der Geer e~ al., 1995). Using peptide binding studies with
mutant peptides, the present illV~;llLUl:~ characterized the nature of the PTB-binding site. The
presence of an A.sn residue three residues amino-tprmin~l to the P.Tyr appears to be
5 absolutely essential for binding to the PTB domain. In coLlLl~L, the Pro appears to be
dispensable for binding to the PTB domain in vitro. Addition of the Pro increases the
affinity and this may be important for binding in vivo, consistent with the observation that
the Pro appears to be conserved in many Shc PTB-binding sites.
It was found that the activated insulin receptor, which also has an
2 o autophosphorylation site c~-nt~ined 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, however,
becomes phosphorylated in response to insuiin 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
2 5 residue five or six residue amino-tPnnin~l to the P.Tyr is hllpol ~ for high affinity binding
by the Shc PTB domain. A phosphopeptide with two Ala residues at these positions still
binds to the Shc Pl'B domain but with an affinity that is aJ~pl~)~illlately 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-t~rmin~l to the P.Tyr disrupts high affinity binding
3 o completely. A peptide, derived from the insulin 1 ecep~or, 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. Conversely, the ability to bind
the Shc PTB domain was destroyed by a single amino acid change outside the Asn-Pro-X-
~ P.Tyr motif in an NGF rec~Lur derived phosphopeptide (Table 1). It appears that different
3 5 PTB domains all recognize Asn-Pro-X-P.Tyr or Asn-X-X-P.Tyr and that further specificity
results from interactions of the PTB domain with amino acid residues outside this
recognition motif. Furthermore, the presence of particular residues at certain positions
CA 0223~7~6 1998-04-24
WO 97/lS318 PCT/US96/1708(~
-- 22
within the binding site could prohibit certain PTB domains from binding without affecting
the binding of other PTB domains. This is partially illustrated by the observation that the
presence of a Ser five residues amino-tprmin~l to the P.Tyr prevents binding of the Shc PTB
domain. An understanding of PTB-binding specificity enables accurate predictions to be
5 made as to which proteins will bind to particular PTB-cont~ining adaptor or .ci~n~llin~
molecules. In~ ition~itenablesmanipulationof the.~t;l~ eofPTBdomain-cont~ining
pLo~eills that are ~~c uiled by growth factor receptors without rh~n~ing the actual phosphate
acceptor sites. For in.ct~ncç, pho~l,ho.ylation of both the insulin receptor substrate 1 (IRS-l)
and Shc appears to depend on a low affinity interaction with the insulin receptor at Tyr 960
0 (Backer et al., 1990; White et al., 1988; Yonezawa et al., 1994). By r.h~nging residues
amino-t~o.rmin~l of the Asn-Pro-X-P.Tyr motif it may be possible to abolish specifically
phosphorylation of either one of these polypeptides by the insulin receptor. Conversely, it
may be possible to create an insulin receptor that interacts much stronger with either Shc
or IRS-1.
Several Arg residues that are conserved in SH2 domains have been shown to be
directly involved in P.Tyr binding (Pawson, 1995; Pawson and Gish, 1992). Based on its
fimrti~n~l homology with the SH2 domain further characterization of the PTB domain by
mutagenesis of Arg residues that are conserved in the PTB domains of dirreLell~ members
of the Shc family has been carried out. The FLVRES sequence (Phe-Leu-Val-Arg-Glu-Ser)
2 o has been conserved between SH2 domains with the Arg being the only invariant residue
present in all SH2 ~lom~inc described thus far ~Pawson, 1995; Pawson and Gish, 1992). An
Arg residue present within the sequence YLVRYM (Tyr-Leu-Val-Arg-Tyr-Met) (residues
52-57), possibly representing a rudimentary FLVRES motif in the Shc PTB domain, was
mutated without an effect on its ligand-binding abilities; mutation of this residue in SH2
25 clom~inc destroys their ability to bind to phosphotyrosine (Marengere and Pawson, 1992;
Mayer et al., 1992). This is consistent with the notion that PTB and SH2 domains are
structurally unrelated. The studies described herein have defined an Arg residue in the
carboxy-t~rminll~ of the PTB domain that is important for its interaction with activated
growth factor receptors. This Arg residue is conserved in dShc and its presence was found
3 o to be Pc~-o.nti~l for binding of the dShc PTB domain to DER, the drosophila homolog of the
EGF receptor. Thus the need for this Arg residue for PTB-ligand interaction has been
conserved in evolution between drosophila and man.
As indicated earlier, Shc appears to be important for signal transduction downstream
of growth factor and cytokine receptors (Burns et al., 1993; Crowe ef al., 1994; Cutler ef
35 al., 1993;Lanfranconeetal., 1995;Peliccietal., 1992;Pronketal., 1993;Ravichandran
etal., 1993; Segattoetal, 1993;Yokoteetal., 1994). ThereisevidencethatShcmaybe
involved in Ras activation pr~sumably throug~ its interaction with Grb2 and Sos (Buday and
CA 0223~7~6 1998-04-24
WO 97/15318 PCT/US96~17~18a
-- 23
Duwllw~.l, 1993; Crowe et al., 1994; Egan et al., 1993; Gale et al., 1993; Li et al., 1993;
Myers ef al., 1994; Rozakis-Adcock et al., 1993; Rozakis-Adcock et al., 1992; Salcini et
al., 1994; Sasaoka etal., 1994).
~ mrle 2
The ability of the peptides listed in Table 2 to inhibit the binding of human Shc
PTB domains to activated EGF-receptor (R) or NGF-R (Trk) was investigated. The
following materials and methods were used in the assays:
Peptides. The peptides are listed in Table 2.In Table 2 the d~ign~ti~n "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
10 is ~;y~ d by a ~ lfide bond between two cysteines on each of the N- and C-termini;"P"
refers to peptides which have penetrating sequences on the N-t~rmiml~ where P-l 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
15 (Appligene) and CG~IENPQPYFSD.
Fusion ~ GST-ShcB and GST-R175M fusion proteins were pl~dl~d as described
in van der Geer et al., 1995.
In vitro binding assay. HER14 cells (3T3 cells ~ essing EGF-R) were starved in 0.5%
CS media for 24 hours and stimlll~ted with 100 ng/ml EGF for 5 min. Cells were Iysed and
2 0 mixed with GST, GST-ShcB or GST-R175M beads. Proteins which bound to beads were
resolved on SDS-PAGE and ~l~tected by anti-phospho-Tyr antibody (4G10) or by anti-EGF-
R.
Results:
Tnhihi~ion of 1~ by Peptides In Vitro. The peptides listed in Table 2 were added to
25 cell Iysates at 5 ,uM, incubated for 30 min., and treated with GST-ShcB beads. Proteins
bound to GST-ShcB were detecte.d by anti-phospho-Tyr antibody. A peptide having the
Shc PTB binding motif of Trk inhibited the association of Shc and EGF-R while a peptide
with an amino acid substitution from Asn to Ala (Trk N to A), or an IRS-l binding site of
insulin receptor (Ins) did not inhibit (Figure 8, Panel A). The peptide designated C-2
3 o inhibited the binding of Shc and EGF-R completely in vitro (Figure 8, Panels A, B). P-2,
dissolved in Hepes buffer and precipi~ d, showed weak inhibition (Figure 8, Panel B). The
experiment was rep~eated with the peptides dissolved in DMSO (Figure 9). P-2 dissolved in
DMSO solution showed strong inhibition at 5 ~M; C-2 inhibited complex formation as
- described previously; and P-l, which is not phosphorylated on Tyr residues did not inhibit
3 5 the association of Shc and EGF-R at 100 IlM.
The dose responses of the peptides was inv~-sti~t~od using the in vitro binding assay.
While C-2 showed strong inhibition at 5 !lM, C-3, C-4, and C-5, did not inhibit (Figure 10,
CA 0223~7~6 1998-04-24
WO 97/15318 PCT/US96/17080
-- 24
Panel A). C-l exhibited about 50% inhibition at lOO~lM (Figure 10, Panel A). P-2(dissolved in Hepes buffer) at a concentration of lOO,uM plGvenlGd the association of
Shc/EGF-R completely, and it showed slight inhibition at 511M compared to the negative
control (P-l) (Figure 10, Panel B). P-2 dissolved in DMSO showed strong inhibition at 5
5 IlM while P-2 at 0.5~1M did not block the protein interaction (Figure 10, Panel C).
Peptide localization in cells. To examine peptide loc~li7~ti- n, cells were treated with P-l
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 0.1511m section from the top of the cells to the bottom. The image analysis
1 0 of cell staining demonstrated that the P-2 peptide localized in the cytoplasm of cells, and
not in the nucleus. Cells treated with P-l peptide were not stained by anti-phospho-Tyr
antibody, confirmin~ the specifity of the immllnnfluoroscence st~ining In a time course
analysis, cells were serum-starved for 24 hours, and incubated with peptide (5 ~4M) for
various time periods. Cells were stained by anti-phosphoro-Tyr antibody and analyzed
15 under an immlln~ oroscence microscope. Cells treated with P-2 peptide retained phospho-
peptide for up to 24 hrs . In a dose response experiment, cells were starved in serum-free
medium for 24 hours, cultured with peptide at various concentrations, and stained with anti-
phospho-tyrosine antibody (4G10) to detect the peptide c~nt~ininp phospho-tyrosine residue
(red) and by Hoechst 33258 for nuclei (blue). P-2 peptide was detected at 0.5,u M and 1
2 o ,u;M in cells . No signals were detected with P-1 peptide in concentraions up to l,u M and
weak non-specific staining was observed at 5,uM.
Inhibition of PTB function in vivo. Growth inhibition of cells.
HER14 cells were starved for 24 hours (Figure l l, Panel a), or 48 hours (Figure 11,
Panel b), treated with P-l or P-2 peptide for 2 hrs and stimulated with l OOng/ml EGF. Cell
2 5 proliferation was measured by3H-TdR uptake (Figure 11). When cells were starved for 24
hrs, P-1 and P-2 peptides slightly inhibited the proliferation of cells compared to the
positive control i.e. EGF alone (a). In cells starved for 48 hrs, both peptides m~rkeflly
plGvGllted cell proliferation. In particular, about 84% inhibition was observed at 1.25,u M.
Both the P-l and P-2 peptide demonstrated inhibitory activity, suggesting a non-specific
3 0 effect was induced by adding peptides. The effect may be due to the internalphosphorylation of P-l peptide by activated kinase(s) after growth factor stim~ tion. The
above experiments were repeated with cells which were serum starved for 24 hrs. Cells
plGLlG~led with P-1 or P-2 did not show any decrease of cell growth rate when compared to
EGl? treated cells. Cells plGLlG~LGd with C-2 inhibited cell growth roughly in a dose
35 dependent manner (Figure 12). The experiment was repeated using C-l peptide as a
negative control, and C-2 did not inhibit cell growth (Figure 13).
A non-Hodgkin's lymphoma cell line, SupM2 was used in cell proliferation assays
CA 0223~7~6 1998-04-24
WO 97/153~8 PCT/US96/17080
-- 25
as described above. Supl~2 has a chromosomal translocation, r~eultin~ in the ~ ession
of a fusion protein of Alk and Npm. The Al~/Npm fusion protein has a motif which is
,e.;Led to be a Shc PTB binding domain, and the cell proliferation of SupM2is believed
- to be dependent on thle Shc paLw~y. SupM2 cells in the indicated cell number (Figure 14,
5 Panel a) or at a conce.lLl~Liol~ of 2 x1 04/well (Figure 14, Panel b), were grown in serum-free
RPMI 1640 medium for 24 hr, treated with peptide for 2 hrs, then, stim~ ted with 20%
FBS overnight, and moni~Jlt;d by 3H-tdR pulse. P-1 and P-2 peptides in_ibited cell growth
to background level (Figure 14), and cell proliferation was completely inhibited at 70nM.
To examine the O~ UIll dose of peptides, serial ~ tinn~ of peptides were used in thle
10 assay. At 60pM, both peptides .,u~lessed thle cell growth completely.
Inhibition of PT]B function in vivo. MAPK activation.
Thle phnsphlnrylation state of MAPK was e~mined after treatment wit_ t_e peptides.
PC12 cells were treated with peptide, stimlll~ted with 50nglml NGF, and a cell lysate was
pl~aled. Pl~leh1s were resolved on acrylamide gel to separate phosphorylated- and non-
15 phosphnrylated MAPK. Erk-1 was detected by Westem blotting. Figure 15, Panel a shows
the series of samples treated with P-1 or P-2, and Panel b shows the samples treated with
C-1 or C-2. When cells were treated with NGF, the bands expected for Erk-1 and Erk-2
were slightly shifted and showed higher molecular weights, demonstrating phosphorylation
of Erk-1 and Erk-2. However, no inhibition was detected in the group treated with P-2 or
2 o C-2. To confirm this result, cell lysates of PC 12 described above were imml-noprecipiLal~d
with anti-Erk-1 antibody, and the proteins were detected by anti-phospho-Tyr antibody
(Figure 16). In Figure 16, Panel a shows sarnples treated with P-1 or P-2, and Panel b
shows samples treated with C-l or C-2. Two major bands were detected with predicted
molecular weights of about 47kDa and about 42kDa, respectively. A weak band of 47kDa
25 was detected in a sample treated with plei.~,.."lne serum. Therefore, pp47 appeared to be
a non-specific protein whereas pp42 is phosphorylated Erk-l. No differences in
phosphnrylation state of Erk-1 were observed among the groups treated with the peptides.
The above described experiment was repeated using 3T3Trk cells stimlll~ted with
NGF. The results were similar to that obtained with PC12 cells. No specific inhibition of
3 o Erk-1 and Erk-2 phosphorylation by C-2 or P-2 was observed.
In sllmm~ry, the efficacy of the peptides in Table 2 were assayed for the ability to
- inhibit the interaction between the Shc PTB domain and growth factor receptors. P-2 and
C-2 peptides dem~nstrated strong inhibitory activities in in vitro binding assays. P-2 was
found to localize in the cell cytoplasm by simply adding the peptide into the culture
35 medium. In prelimin~ry experiments, specific inhibition of cell growth or of MAPK
activation by the peptides was not demon.~ ed in vivo. However, the in vivo assay system
requires further optimization.
CA 0223~7~6 1998-04-24
WO 97/15318 PCT/US96/1708(~
-- 26 --
Having illustrated and described the principles of the invention in a preferred
embodiment, it should be appreciated to those skilled in the art that the invention can be
modified in arrangement and detail without departure from such principles. We claim all
modifications coming within the scope of the following claims.
All publications, patents and patent applications referred to herein are incol~ol~led
by reference in their entirety to the same extent as if each individual publication, patent or
patent application was specifically and individually indicated to be incorporated by
reference in its entirety.
Below full citations are set out for the references referred to in the specification is
10 a listing and detailed legends for the f1gures are provided.
The application contains sequence listings which form part of the application.
CA 02235756 1998-04-24
WO 97/lS318 PCT~US96~17080
-- 27
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K.-M. V. Lai, J. P. Olivier, G. D. Gish, M. Henkemeyer, J. McGlade and T. Pawson (1995)
Mol. Cell. Biol., 15: 4810-4818.
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J. Schlessinger (1993) Nature, 363, 85-88.
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L. E. M. Marengere and T. Pawson (1992) J. Biol. C*em., 267, 22779-22786.
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A. Obermeier, R. A. Bradshaw, K. Seedorf, A. Choidas, J. Schlessinger and A. Ullrich
(1994) E~BO J., 13, 1585-1590.
Y. Okabayashi, Y. Kido, T. Okutani, Y. Sugimoto, K. S~k~ .hi and M. Kasuga (1994) J.
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T. Pawson and G. D. Gish (1992) Cell, 71, 359-362.
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20 R. A. Roth, D. J. Cassell, K. Y. Wong, B. A. Maddux and I. D. Goldfine (1982) Proc. Na~l.
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CA 02235756 1998-04-24
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- 29 -
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CA 0223~7~6 1998-04-24
WO 97/lS318 PCT/US96/17080
-- 30 --
DETAILED FIGURE LEGENDS
Figure lA and Figure lB. The Asn present within the Asn-Pro-X-P.Tyr motif is
essential for binding to the PTB domain. Figure lA. GST (lane 2) and GST Shc PTB(lanes 1, 3-6) fusion proteins bound to glutathione-agarose were incubated with NGF
5 receptors present in Iysates from control (lane 1) and NGF-stim~ ted (lanes 2-7) cells in
the absence (lane 1-3) and presence of Wt (lane 4) and mutant (lanes 5 and 6) competing
P.Tyr cont~ining peptides, based on the sequence around Tyr 490 the Shc PTB domain
binding site in the NGF receptor. Bound proteins were analyzed by anti-P.Tyr
immun~blotting. Competing peptides, Wt NGF l~cel~r (Wt NGFR): His-Ile-Ile-Glu-Asn-
10 Pro-Gln-P.Tyr-Phe-Ser-Asp (lane 4); NGF recel)lur 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 lB. The blot
shown under A was stripped and reprobed with an antiserum raised against the NGFreceptor.
15 Figure 2. Sul~.,lilulion of either the Asn or the Pro in the PTB-binding site affects its
ability to bind to the PTB domain. Surface plasmon resonance technology was used to
test the ability of Wt and mutant phosphopeptides, based on the sequence around Tyr 490
the Shc-binding site in the NGF l~c~L~l for their ability to compete for binding of the Gst-
Shc PTB domain fusion protein to the immobilized polyoma middle T antigen peptide (L-S-
2 0 L-L-S-N-P-T-P.Y-S-V-M-R-S-K). Wt, 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( ~ ).
Figure 3. P. ~_.~ce of a Asn-Pro-X-P.Tyr motif in the ju~ta membrane domains of the
NGF and insulin rec~l,tors. Both the NGF receptor and the insulin receptor contain an
autophosphorylation site witnin an Asn-Pro-X-P.Tyr motif in the juxta membrane domain,
25 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.
Figure 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
3 0 1, 2, 5, 6, 9, and 10) from control (lanes 1, 5, and 9) and growth factor-stim~ ted (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) were analyzed by anti-P.Tyr immlm~blotting. Anti-NGF receptor
(lanes 3, 4, 7, and 8) and anti-insulin receptor immunopreci~,ikites (lanes 11 and 12) from
35 control (lanes 3, 7, and 11) and growth factor stimlll~ted (lanes 4, 8, and 12) were analyzed
in parallel. Figure 4B. Wt (lanes 1 and 2) and Phe 490 mutant (5 and 6) NG~ receptors
present in Iysates from control (lanes 1 and 5) and NGF-stimlll~ted (lanes 2 and 6) cells
CA 0223=,7=,6 1998-04-24
WO 97/15318 PCTAUS96/17080
- 31 -
C,~ SSillg Wt (NGFR) orPhe 490 mutant (F490NGFR) and insulin receptors (IR) present
in Iysates from control (lane 9) and insulin-stim~ ted (lane 10) cells were incubated with
GST-Shc PTB fusIon pluleills bound to gl~t~t1~ion~-agarose~ Bound ~loleins were analyzed
~ by anti-P.Tyr immlln~blotting. Anti-NGF receptor immlln~lprecipi~Les (lanes 3, 4, 7, and
5 8) and anti-insulin l~ce~or immunoplt:cipi~es (lanes 11 and 12) from control (lanes 3, 7,
~ and 11) and growth factor-stimul~t~d (lanes 4, 8, and 12) cells were analyzed in parallel.
Figure SA and Figure 5B. An ~ ~tic residue five or six amino acids amino-terminal
to the P.Tyr is an important d~ .ant for Shc PTB binding. Figure 5A. GST-Shc
PTB domain fusion proteins bound to glutathione-agarose were incubated with activated
0 NGF receptors present in lysates of NGF-stimlll~ted cells in the absence (lane 1) or presence
(lanes Z-7) of 2~1M competing Wt and mutant phosphotyrosine cont~ining 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 rec~lur (lanes 6 and 7). Wt NGF l~c~r peptide (Wt-NGFR, lane 2): H-I-I-
5 E-N-P-Q-p.Y-F-S-D; Ala-6 NGF lt;Ct~Lul peptide (NGFR-HAI): H-A-I-E-N-P-Q-p.Y-F-S-
D; Ala-6, Ala-5 NGF l~:ce~ol 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
ll;ceplor 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
2 0 5B. Phosphopeptides based on the sequence around Tyr 490, the Shc-binding site in the
NGF recel~lur ~H-I-I-E-N-P-Q-p.Y-F-S-D(-)) or Tyr 960 in the insulin receptor (Y-A-S-S-
N-P-E-p.Y-L-S-A (O)) and substitutions at position -5 and -6 with respect to the P.Tyr in
the NGF receptor peptides ~H-A-S-E-N-P-Q-p.Y-F-S-D(-)) and the insulin receptor peptide
(Y-A-I-S-N-P-E-p.Y-L-S-A(~)) were tested by surface plasmon resonance analysis
2 5 technology for their ability to compete for the binding of the GST-Shc PTB domain to the
immobilized polyoma middle T antigen peptide (L-S-L-L-S-N-P-T-p.Y-S-V-M-R-S-K).
Figure 6A and Figure 6B. The re~ I for an Arg residue at position 17~ in the
human Shc PTB domain has been conserved in evolution. Figure 6A. GST fusion
proteins cont~inin~ Wt (lanes 1 and 2) or mutant (lanes 3-11) Shc PTB domains were
3 o incubated with NGF receptors present in lysates of conkol (lane 1) and NGF-stim~ ted
cells (lanes 2-11). Bound proteins were analyzed by anti-P.Tyr blotting. Figure 6B. Human
~ EGF receptors bound to GST fusion proteins c~",l;~;.,;"g Wt (lanes 1 and 2) or Met 175 (lane
3) and Lys 175 (lane 4) mutant human Shc PTB dom~in~ in lysates from control (lane 1)
or EGF-stim~ ted cells (lanes 2-4) were analyzed by anti-P.Tyr blotting. In parallel GST
35 (lane 8) and GST fusion proteins cont~inin~ Wt (lane 7) or an Ala 151 mutant (lane 9)
drosophila Shc PTB domain bound to glutathione-agarose, were incubated with fly lysates
c~ g activated Torso-DER chimeric proteins that contain the cytoplasmic domain of
CA 0223~7~6 1998-04-24
WO 97/15318 PCT/US96/17080
-- 32
DER; bound proteins were detected by anti-P.Tyr blotting. An anti-Shc ~lane 5) and a
normal rabbit serum imml-n~precipitate (lane 6) from the same fly lysates are shown as
controls.
Figure 8. Peptides Competition in In Vitro Binding Assay. Cell Lysates were pre-5 incubated with 5~M of a~p,upliate 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-I binding domain on insulin-R.
Figure 9. Cc pe~ on Assay of P~ .g Peptide. Cell lysates were pre-treated with
appropriate peptides. Proteins were precipitated by GST-ShcB and detected by anti-
0 phospho-tyrosine antibody. Peptides were prepared in DMSO solution.
Figure 10. Dose-Response Analysis of Peptides in in vitro R:n(ling Assay. Cell Lysates
were prepared and inc~lb~tecl with applupliate 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-l and P-2 peptides were dissolved in Hepes
5 buffer (B), and in DMSO solution (C).
Figure 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 applup.iate peptide for
2 hrs, then stim~ ted with lOOng/ml EGF overnight. Cell proliferation was monitored by
3H-TdR uptake.
20 Figure 12. Proliferation of ~:ER14 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 stimlll~tion. Cell proliferation was induced with
l OOng/ml EGF overnight, then monitored by 3H-TdR uptake.
Figure 13. Pr~,lir~. ~lion of ~R14 cells treated with cyclic peptides. Cells were starved
25 for 48 hrs in serum-free D-MEM medium. Appropriate peptide was added in various
concentrations and cultured for 2 hrs prior to EGF stimul~tinn. Cell proliferation was
in~ ced with lOOng/ml EGF overnight and monitored by 3H-TdR uptake.
Figure 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
3 o 2xlO4/well (b). Cells were treated with penetrating peptide in various concentrations for 2
hrs prior to cell stimulation. Cell proliferation was in~luced with 20% FPS overnight and
monitored by 3H-TdR pulse.
Figure 15. MAPK Activation on PC12 Cells Treated with Peptides. PC12 cells were
treated with a~c.upliate peptide at various concentrations. Cells were stim~ ted with
3 5 50ng/ml NGF for 5 min., then cell lysates were prepared by standard methods. Each lane
contains 10,ug protein and MAPK (Erk-l) was detected by anti-Erk-l polyclonal Ab.
Arrows represent activated Erk-1/2 (b).
CA 02235756 l998-04-24
WO 97/15318 PCT/US96/17080
-- 33
Figure 16. Dct~ of A~lival_d MAPK on PC12 Cells Treated with Peptides. PC12
cells were treated with peptides for 4 hrs prior to stim~ tion. Cells were stim~ ted with
50ng/ml NGF for S min. and cell lysates were prepared according to standard methods.
~ SOO,ug of cell lysates were immun-)precipitated with anti-Erk-l polyclonal antibody, and
5 activated Erk-l proteins were detected by Western blotting of anti-phospho-tyrosine
antibody (4G10). Arrow represents activated (phosphorylated) Erk-l and asterisk shows
non-specific band.
CA 02235756 1998-04-24
WO 97/15318 PCT/US96tl7080
TABLE 1
Phosphotyrosine-contAlnin~ peptides IC SC
H-I-I-E-N-P-Q-P.Y-F-S-D 175 nM
H-I-I-E-~-P-Q-P.Y-F-S-D 80000 nM
H-I-I-E-N-A-Q-P.Y-F-S-D 2500 nM
H-A-I-E-N-P-Q-P.Y-F-S-D 20 nM
H-I-A-E-N-P-Q-P.Y-F-S-D 250 nM
H-A-A-E-N-P-Q-P.Y-F-S-D 475 nM
H-A-S-E-N-P-Q-P.Y-F-S-D 15000 nM
Y-A-S-S-N-P-E-P.Y-L-S-A 7000 nM
Y-A-I-S-N-P-E-P.Y-L-S-A 90 nM
Table 1. Peptide competition of the GST-Shc PTB domain binding to a polyoma
middle T antigen phosphopeptide (L-S-L-L-S-N-P-T-P.Y-S-V-M-R-S-K). Surface
plasmon resonance technology was used to evaluate the ability of phosphopeptidesderived from sequences around the Tyr 490, ~e Shc binding site in the NGF
receptor (H-I-I-E-N-P-Q-P.Y-F-S-D) and Tyr 960 in the insulin receptor (Y-A-S-S-N-P-
E-P.Y-L-S-A) to bind to the Shc PTB domain. Arnino acid substitutions were
introduced into the peptides (shown in bold). Peptide concentrations that inhibit
binding by 50% (1C50) are listed.
CA 02235756 1998-04-24
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WO 97/15318
-- 35 --
TABLE 2
Code Amino aGid sequen~e
Trk wild type HiIENPQpYFSiD 175nM
IENPQpYFSi~ 4,uM
G 1 t~yclo~ NP~pYF~PG)
C-~ CL~NPQ~CF
SS
~3 C:y~lo-(NPQpY)
¢.4 Cy¢Io-(NPQpYG)
~5 Cyclo (NP~p~f~G~
P.1 . R~lKIW~Ql~ MiK~VKK-iHlIl~NPQYFSD
P-2 RC~lKlWFC:lNi~RMKWKK~WIlENPapYFSD
P.3 Blotin-At::A-MVALLPAVLlLALLAP-HlIENPQYFSD
p.4 Biotin-A~A-MvALLpAvL~ALLAp~ ENpQpyFsD
p s ~BHIIENPC;~pY-S~)
SS--BlotIn~Basic pen~ in~ psptide
~RQIKIWFQNRRMKWKK~)
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WO 97/15318 PCT~US96/17080
- 36 -
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS:
(A) NAME: Mount Sinai Hospital Corporation
(B) STREET: 600 University Avenue, Suite 970
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE: M5G lX5
(G) TELEPHONE NO.: (416) 586-3235
(H) TELEFAX NO.: (416) 586-3110
(A) NAME: Asahi Chemical Industry Co., Ltd.
(B) STREET: Hibiya-Mitsui Bldg., 1 - 2, Yuraku-cho,
1-Chome, Chiyoda-Ku
(C) CITY: Tokyo
(D) STATE: N/A
(E) COUNTRY: Japan
(F) POSTAL CODE: N/A
(G) TELEPHONE NO.:
(H) TELEFAX NO.:
(A) NAME: van der Greer, Peter
(B) STREET: 33 Wood Street, Apt. 2204
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): M4Y 2P8
(A) NAME: Wiley, Sandra
(B) STREET: 33 Wood Street, Apt. 2204
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): M4Y 2P8
(A) NAME: Gish, Gerald D.
(B) STREET: 6 Grangemill Crescent
(C) CITY: Don Mills
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): M3B 2J2
(A) NAME: Pawson, Tony
(B) STREET: 34 Glenwood Avenue
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): M6P 3C6
(A) NAME: Toma, Kazunori
(B) STREET: 4-8-36, Yokodai
(C) CITY: Sagamihara
(D) STATE: N/A
(E) COUNTRY: Japan
(F) POSTAL CODE: ZIP 229
(ii) TITLE OF INVENTION: Peptides Which Interfere With the
Interaction of a Phosphotyrosine-Binding (PTB) Domain
Cont~;n~ng Protein With a PTB Domain B;n~;n~ Site
(iii) NUMBER OF SEQUENCES: 29
CA 02235756 l998-04-24
WO 97/1~318 PCT~US96/~ 7081
-- 37 --
(iv) CoR~po~n~r~ nD~Cs
~A) ADDRESSEE: HOWSON AND HOWSON
IB) STREET: Spring House Corporate Cntr., P.O. Box 457
,C, CITY: Spring House
D, STATE: Pennsylvania
EI Cc)lJhLKy: USA
~F, ZIP: 19477
(v) COMPUTER ~T.~AnARr.~ FORM:
AI MEDIUM TYPE: Floppy di~k
~B COMPUTER: IBM PC compatible
,C, OPERATING SYSTEM: PC-DOS/MS-DOS
lD, SOFTWARE: PatentIn Relea~e ~1.0, Version ~1.30
(vi) ~u~X~ APPLICATION DATA:
(A) APPLICATION NUM3ER: WO
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) AL~/AGENT INFORMATION:
(A) NAME: Kodroff, Cathy A.
(B) REGISTRATION NUMBER: 33,980
(C) REFERENCE/DOCXET NUMBER: 3153-199
(ix) T~T~cnM~JNIcATIoN INFOR~ATION:
(A) TELEPHONE: (215) 540-9200
(B) TELEFAX: (215) 540-5818
(2) INFOR~ATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
AI LENGTH: ll amino acids
lBI TYPE: amino acid
,C~ STRANDEDNESS: unknown
~D TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATUaE:
(A) NAME/KEY: Peptide
(B) LOCATION: l..11
(D~ OTHER INFORMATION: /note= "Pho~phorylated at Tyr~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
His Ile Ile Glu Asn Pro Gln Tyr Phe Ser As~
l 5 10
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CY~RACTERISTICS:
(A) LENGTH: ll amino acid~
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLCGY: linear
( _i ) M~T ~CJT ~ TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(3) LOCATION: 1..11
(D) OTHER INFORMATION: /note= ~Phosphorylated at Tyr"
CA 0223~7~6 1998-04-24
WO 97/15318 PCT/US96/17080
- 38 --
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
His Ala Ile Glu Asn Pro Gln Tyr Phe Ser Asp
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ll amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: l..ll
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
His Ile Ala Glu Asn Pro Gln Tyr Phe Ser Asp
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ll amino acids
(B) TYPE: amino acid
(C) sTRpNnp~n~ ss unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: l..ll
(D) OTHER INFORMATION: /note= nPhosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Ala
(2) INFORMATION FOR SEQ ID NO:5:
( i ) ~;OU~;N~:~; CHARACTERISTICS:
(A) LENGTH: ll amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: l..ll
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Tyr Ala Ile Ser Asn Pro Glu Tyr Leu Ser Ala
~2) INFORMATION FOR SEQ ID NO:6:
CA 0223~7~6 l998-04-24
W O 97J15318 PCTAUS96~I708~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
tB) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..11
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Ser Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A'l LENGTH: 12 amino acids
(B TYPE: amino acid
(Cl STRANDEDNESS: unknown
(Dl TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..11
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Thr Trp Ile Glu Asn Lys Leu Tyr Gly Thr Ser Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE:-amino acid
(C) STRAWDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Leu Leu Leu Ser Asn Pro Ala Tyr Arg Leu Leu Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
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- 40 -
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Tyr Ala Ser Ser Asn Pro Glu Tyr Leu Ser Ala Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) ~Uu~ DESCRIPTION: SEQ ID NO:10:
Val Ser Val Asp Asn Pro Glu Tyr Leu Leu Asn Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) sTRAMn~nN~ss: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ser Leu heu Ser Asn Pro Thr Tyr Ser Val Met Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO:12:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
CA 0223~7~6 l998-04-24
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Asn Glu Met Ile Asn Pro Asn Tyr Ile Gly Met Gly
1 5 10
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Glu Met Phe Glu Asn Pro Leu Tyr Gly Ser Val Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..7
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Ile Glu Asn Pro Gln Tyr Phe
1 5
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..8
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ile Glu Asn Pro Gln Tyr Phe Ser
1 5
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- 42 -
(2) INFORMATION FOR SEQ ID-NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..7
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Ala Glu Asn Pro Gln Tyr Phe
1 5
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..8
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ile Glu Asn Pro Gln Tyr Phe Ser Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..7
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Ile Ser Asn Pro Glu Tyr Leu
1 5
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
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- 43 -
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
tix) FEATURE:
(A) NAME/~EY: Peptide
(B) LOCATION: l..ll
(D) OTHER INFORMATION: /note= nPhosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Val Leu Ala Asp Asn Pro Ala Tyr Arg Ser Ala
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 14 amino acids
B) TYPE: amino acid
C) STRANDEDNESS: unknown
~D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: l..14
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Ala Leu Leu Leu Ser Asn Pro Ala Tyr Arg Leu Leu Leu Ala
l 5 lO
(2) INFORMATION FOR SEQ ID NO:2l:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 18 amino acids
B) TYPE: amino acid
C) STRANDEDNESS: unknown
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/~EY: Peptide
(B) LOCAT]:ON: l..18
(D) OTHER INFORMATION: /note= nPhosphorylated at Tyr ll"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Gly Pro Leu Tyr Ala Ser Ser Asn Pro Glu Tyr Leu Ser Ala Ser Asp
l 5 l0 15
Val Phe
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
~ I'A) LENGTH: 15 amino acids
~B) TYPE: amino acid
~C) STRANDEDNESS: unknown
~D) TOPOLOGY: linear
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- 44 -
(ii) MOLECULE TYPE: peptide
~ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Pro Val Ser Val Asp Asn Pro Glu Tyr Leu Leu Asn Ala Gln Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID No:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STR~Mn~nN~..~S: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID No:23:
Leu Ser Leu Leu Ser Asn Pro Thr Tyr Ser Val Met Arg Ser Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID No:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..18
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Val Ser Ser Leu Asn Glu Met Ile Asn Pro Asn Tyr Ile Gly Met Gly
1 5 10 15
Pro Phe
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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- 45 -
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Leu Leu Leu Thr Lys Pro Glu Met Phe Glu Asn Pro Leu Tyr Gly Ser
1 5 10 15
Val Ser Ser Phe
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STR~MnEnN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "cyclic"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..10
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Ile Glu Asn Pro Gln Tyr Phe Ser Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID No:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) sTRAMn~n~F.~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TY~E: other nucleic acid
(A) DESCRIPTION: /desc = "cyclic"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..13
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(Xi) ~OU~N~ DESCRIPTION: SEQ ID NO:27:
Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Ala Pro Gly
1 5 10
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "cyclic"
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- 46 -
(ix) FEATURE:
(A) NA~E/KEY: Peptide
(B) LOCATION: l..14
(D) OTHER INFORMATION: /note= "Phosphorylated at Tyr"
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:28:
His Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Ala Pro Gly
l 5 l0
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQuEN-cE CHARACTERISTICS:
(A) LENGTH: l0 amino acids
(B) TYPE: amino acid
(C) sTRANn~n~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "cyclic"
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: l..l0
(D) OTHER INFORMATION: /note= nPhosphorylated at Tyr"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Cys Ile Ile Glu Asn Pro Gln Tyr Phe Cys
l 5 l0
