U.S. patent application number 11/893643 was filed with the patent office on 2009-02-12 for peptides and chemical compound for inhibition of shp2 function.
Invention is credited to Yehenew Mekonnen Agazie, Peter Mico Gannett.
Application Number | 20090042788 11/893643 |
Document ID | / |
Family ID | 38877438 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090042788 |
Kind Code |
A1 |
Agazie; Yehenew Mekonnen ;
et al. |
February 12, 2009 |
Peptides and chemical compound for inhibition of SHP2 function
Abstract
The present invention relates to the inhibition of the function
of SHP2 by both anti-SHP2 peptides and the chemical compound
4-(2-sulfaminoethyl)benzoic acid, SEBA, and SEBA derivatives
binding to the phosphotyrosyl phosphatase domain of SHP2 thereby
inhibiting the function of SHP2 both in vitro and in vivo. In
addition, the inhibition of SHP2 may be useful as a treatment for
human disease, and it has been shown that interfering with SHP2
function using the anti-SHP2 peptides and SEBA compounds reverses
cell transformation and induces remission of preformed tumors in
vivo demonstrating a possible treatment for cancer.
Inventors: |
Agazie; Yehenew Mekonnen;
(Morgantown, WV) ; Gannett; Peter Mico;
(Morgantown, WV) |
Correspondence
Address: |
WEST VIRGINIA UNIVERSITY RESEARCH CORPORATION
886 CHESTNUT RIDGE ROAD, P.O. BOX 6224
MORGANTOWN
WV
26506-6224
US
|
Family ID: |
38877438 |
Appl. No.: |
11/893643 |
Filed: |
August 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11480814 |
Jul 3, 2006 |
|
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11893643 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
C07K 5/1021 20130101;
C07K 7/06 20130101; A61P 43/00 20180101; C07K 5/0819 20130101; C07K
5/06113 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61P 43/00 20060101 A61P043/00 |
Claims
1. A SEBA derivative comprised of the structure ##STR00002##
wherein R' is selected from the functional group consisting of the
amino acids sequence D-V; D-G; D-A; D-G-D-G; D-A-D-A; D-V-D-V;
D-A-D-V; D-A-D-G; D-G-D-V; D-G-D-A; D-V-D-G; and D-V-D-A, and R is
selected from the functional groups acetate, phosphate, sulfate,
and aminosulfate.
2. A method to inhibit SHP2 function comprising a blocker
preventing the binding of biological substrates to the active site
of the phosphotyrosyl phosphatase domain of SHP2 wherein said
blocker is a SEBA derivative comprising of ##STR00003## wherein R'
is selected from the functional group consisting of the amino acids
sequence D-V; D-G; D-A; D-G-D-G; D-A-D-A; D-V-D-V; D-A-D-V;
D-A-D-G; D-G-D-V; D-G-D-A; D-V-D-G; and D-V-D-A, and R is selected
from the functional groups acetate; phosphate, sulfate, and
aminosulfate binding to said site.
3. The method to inhibit SHP2 function of claim 2 wherein an
effective amount of said chemical compound is used in the treatment
of a human disease.
4. The method to inhibit SHP2 function of claim 3 wherein said
human disease is cancer.
5. A chemical compound SEBA comprising 4-(2-sulfaminoethyl)benzoic
acid.
6. The chemical compound SEBA of claim 5 wherein an effective
amount of said SEBA inhibits the function of SHP2 by binding to the
active site of the phosphotyrosyl phosphatase domain of said
SHP2.
7. The chemical compound SEBA of claim 6 wherein an effective
amount of said SEBA is used in the treatment of a human
disease.
8. The chemical compound SEBA of claim 7 wherein said human disease
is cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Not Applicable
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable
REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM
LISTING COMPACT DISC APPENDIX
[0003] This application contains a Sequence Listing submitted as
follows. The following sequence listing is incorporated by
reference herein in its entirety and is identical to the sequence
listing contained in the body of the application and contains no
new matter.
TABLE-US-00001 SEQ ID: 1 LENGTH: 5 SEQUENCE: Asp-Ala-Asp-Val-Tyr
SEQ ID: 2 LENGTH: 5 SEQUENCE: Asp-Ala-Asp-Gly-Tyr SEQ ID: 3 LENGTH:
5 SEQUENCE: Glu-Ala-Asp-Val-Tyr SEQ ID: 4 LENGTH: 3 SEQUENCE:
Asp-Val-Tyr SEQ ID: 5 LENGTH: 3 SEQUENCE: Asp-Gly-Tyr
BACKGROUND OF THE INVENTION
[0004] 1. Field of Invention
[0005] This invention relates to the inhibition of the function of
SHP2 by anti-SHP2 peptides, the chemical compounds
4-(2-sulfaminoethyl)benzoic acid ("SEBA"), and SEBA derivatives
binding to the phosphotyrosyl phosphatase domain of SHP2.
[0006] 2. Description of the Prior Art
[0007] Protein Tyr phosphorylation and dephosphorylation reactions
play major role in the transduction of growth factor signals from
the surface to the interior of the cell. Phosphorylation involves
addition of a phosphate moiety to the hydroxyl group of the Tyr
residue in proteins or peptides, while dephosphorylation refers to
the removal of such phosphate from the phosphotyrosine.
Phosphorylation reactions are catalyzed by Tyr kinases, while
dephosphorylation reactions are catalyzed by phosphotyrosyl
phosphatases ("PTPs"). Depending on their location in the cell, Tyr
kinases are generally classified as cytoplasmic Tyr kinases
("CTKs") or receptor Tyr kinases ("RTKs"). Most commonly cited CTKs
include Src, focal adhesion kinase ("FAK") and janus kinases
("JAKs") (17, 19, 47, 72, 73), while the most commonly studied RTKs
are the epidermal growth factor receptor ("EGFR") family, the
platelet growth factor receptor ("PDGFR") family, the fibroblast
growth factor receptor ("FGFR") family and the insulin receptor
(30, 45, 82, 96). Similarly, PTPs are classified as cytoplasmic or
receptor type depending on their location in the cell (7, 8, 66,
93). This invention focuses on SHP2, the cytoplasmic PTP that has
been shown to positively modulate RTK signaling, particularly,
signaling by the epidermal growth factor receptor ("EGFR")
family.
[0008] SHP2 possesses two tandemly-arranged Src homology 2 ("SH2")
domains in the N-terminal region and a PTP domain in the C terminal
region (32, 33). Both of these domains are essential for its
biological activity (14, 32, 33, 78, 79, 86). It also possesses two
tyrosine phosphorylation sites and a proline-rich motif (-PXXP-) in
the extreme C-terminal region (10, 29, 69). The SHP2 protein
assumes a "closed conformation" when inactive and an "open
conformation" when active. In the closed conformation, the N-SH2
domain interacts with the PTP domain thus physically impeding the
PTP domain from binding to target substrates. Upon engagement of
the N-SH2 domain with phosphotyrosyl residues, the protein assumes
an open conformation, relieving the PTP domain and rendering the
enzyme active (41). In the N-SH2 domain, Asp61 and Glu76 are the
mediators of this interaction. As a result, mutation of these
residues creates a constitutively active SHP2 (70). Recent findings
show that constitutively active SHP2 mutatants naturally occur in
Noonan Syndrome-associated leukemia (87-89).
[0009] In the catalytic process of most PTPs, including SHP2, Asp
in the WPD loop and the conserved residues in the signature motif
play critical roles in the dephosphorylation reaction (34). In
SHP2, Asp425 acts as a proton donor for the leaving phenolate group
of the substrate and as an acceptor during the hydrolysis of the
cysteinyl phosphate intermediate. The Cys residue conducts a
nucleophilic attack on the phosphate moiety, while the Arg residue
(positively charged) serves as a coordinator of the negatively
charged phosphate group on the substrate. Basically, the Arg
residue mediates substrate binding to the active site of the
enzyme, whereas the Cys and Asp residues catalyze the
dephosphorylation reaction. In addition, Thr466 is essential for
SHP2 catalysis (61), but its specific role has not been
established. Mutation of this residue to Ala provides a new
substrate-trapping mutant of SHP2 (61). SHP2 is a unique PTP that
positively modulates RTK signaling in vertebrates (32, 33, 35, 38).
Interaction of SHP2 with Tyr-phosphorylated RTKs and adaptor
proteins such as Gab1 and 2 and FRS1 and 2 through its SH2 domains
is important for its function (28, 38, 51, 55, 80, 92). This
interaction recruits SHP2 to its substrate microdomain. Deletion of
the N-SH2 domain or mutation of critical residues in the active
site of the enzyme such as Cys459 to Ser or Arg465 to Glu
inactivates SHP2, suggesting that both domains are important for
its function. These mutants have served as dominant-negative
counterparts ("DN-SHP2") in SHP2 studies as their expression
inhibits the activation of the Ras-ERK (ERK for extracellular
signal regulated kinase 1 and 2) and the PI3K-Akt (PI3K for
phosphatidylinositol 3-kinase) signaling pathways downstream of
RTKs (5, 6, 53, 54, 92).
[0010] SHP2 enhances the signaling potential of the EGFR family of
RTKs. The EGFR family comprises four members ("EGFR1-4") and
represents one of the most extensively studied RTKs in the
mammalian system (30, 48, 57, 58, 82, 95). The human homologues are
called HER1, HER2, HER3 and HER4. Because HER1 is commonly known as
EGFR, this abbreviation will be used hereinafter. All family
members are composed of an extracellular ligand-binding region, a
single-pass transmembrane region and a cytoplasmic region
containing a tyrosine kinase domain (except HER3 that has
dysfunctional kinase domain) and tyrosine autophosphorylation
sites. A family of a dozen growth factors, including epidermal
growth factor ("EGF"), transforming growth factor-.alpha.
("TGF.alpha.") and neuregulins, activate these receptors by binding
to the extracellular region (except HER2 that does not require
ligand binding for activation). The binding of a cognate ligand
induces homo- or hetero-dimerization of EGFR molecules leading to
the activation of their tyrosine kinase domain and
autophosphorylation of specific tyrosine residues in the C-terminal
region or Tyr phosphorylation of downstream substrates (24, 26, 28,
43, 51, 59, 71). Phosphorylated tyrosine residues serve as binding
sites for SH2 domain-containing signaling molecules such as the
Grb2-SOS complex, the phosphatidylinositol 3-kinase ("PI3K"), SHP2
and others (74), which leads to formation of multimeric signaling
complexes. At least two known functions are effected by these
interactions: recruitment of enzymes to substrate micro domains
(e.g. Grb2-SOS) and induction of enzyme activity (egs. PI3K, SHP2)
(2, 12, 33, 38, 55, 59, 80, 83). The immediate response to
stimulation with EGF or the related ligands is the activation of
the Ras-ERK (ERK for extracellular regulated kinase 1 and 2) and
the PI3K-Akt (Akt is sometimes referred to as protein kinase B)
signaling pathways, which induce mitogenesis and cell survival,
respectively. Structural and biochemical analyses have shown that
HER2 does not require ligand binding for activation of its tyrosine
kinase domain (36). Thus, HER2 can potentially homodimerize in the
absence of ligand (especially under conditions of overexpression)
or heterodimerize with ligand-stimulated family members. Generally,
HER2 is regarded as the preferred partner of heterodimerization
with the other family members most probably due to the
unconstrained conformation of the dimerization arm of the
extracellular region (36). In case of HER3, heterodimerization with
other family member is the only mechanism for its activation
because it lacks a functional Tyr kinase domain (42).
[0011] Regulation of cell shape and morphology by SHP2 has been
correlated with its effect on actin cytoskeletal reorganization.
Cells expressing dominant-negative SHP2 manifest an increased level
of actin stress fiber formation and assume a flattened morphology,
whereas cells expressing wild-type SHP2 show a low level of actin
stress fiber formation and a more polarized morphology (46, 50,
84). These effects of SHP2 have been ascribed to its negative
regulatory role of the Rho GTPase (25, 52, 84).
[0012] SHP2 suppresses cell adhesion and enhances motility (46, 50,
56, 102). However, the molecular basis underlying these effects is
not clear. Cell migration requires a coordinated cycling between
adhesion and detachment. In a tissue or in confluent monolayer of
cultured cells, movement of a single cell in a defined direction
involves modulation of its interaction with neighboring cells and
the extracellular matrix ("ECM"). Adhesion of cells to the ECM is
mediated by cell surface receptors called integrins. Integrin-ECM
interaction recruits cytoplasmic tyrosine kinases ("CTK") such as
FAK, related focal tyrosine kinase ("RFTK") or Pyk2 and Src (72,
81) to the plasma membrane. Autophosphorylation of FAK initiates
the cascade of tyrosine phosphorylation reactions by recruiting Src
to focal adhesions (72, 81). The adaptor proteins p130.sup.Cas and
paxillin are the other major proteins known to participate in focal
adhesion formations. The SH3 and Src-binding domains of
p130.sup.Cas mediate interaction with FAK and Src, which in turn
mediate Tyr phosphorylation (65, 76). Paxillin possesses
lucine-rich motifs that promote its interaction with FAK and the
other focal adhesion protein vinculin (21, 27). Paxillin also
possesses Tyr phosphorylation sites that mediate SH2 domain
interactions (99). The net effect is the aggregation of integrins,
the formation of multi-protein complexes and the maturation of
focal complexes to focal adhesions (56, 100). SHP2 has been
repeatedly implicated in down regulating focal adhesions, but the
molecular mechanism is not clear.
[0013] SHP2 mediates cell transformation induced by v-Src (39) and
the constitutively active form of fibroblast growth factor receptor
3 (K650E-FGFR3) (6). In K650E-FGFR3-induced transformation,
SHP2-mediated activation of the Ras-ERK and PI3K-Akt pathways was
essential. Recently, it was demonstrated that SHP2 promotes
K650E-FGFR3-induced transformation not only by promoting the
activation of the Ras-ERK and PI3K-Akt pathways, but also by
modulating the interaction of the actin cytoskeleton with adherens
junction (23). The recent discovery of gain-of-function SHP2
mutations in Noonan syndrome and associated leukemia (15, 62, 89)
and the subsequent demonstration of the development of lymphoid
hyperplasia in transgenic mice expressing gain-of-function SHP2
mutants (9, 62) further support the importance of SHP2 in
cancer.
[0014] The driving force behind this invention is that SHP2 is an
essential transducer of cell transformation induced by Tyr kinase
oncogenes, which in turn suggests that it promotes tumor growth.
Recent reports have uncovered molecular mechanisms by which the
phosphatase activity of SHP2 promotes signaling by Tyr kinases,
particularly by the EGFR. The development of a substrate-trapping
mutants of SHP2 was a breakthrough for understanding its molecular
mechanism (4). Substrate trapping refers to the production of
mutant phosphatases that retain substrate-binding ability or
acquire enhanced substrate-binding ability, but are devoid of
enzyme activity (34). Thus, by introducing mutations in the active
site of SHP2, an efficient substrate trapping mutant termed DM-SHP2
(DM for double mutant) was developed (4). Using the DM-SHP2 as a
reagent, the first biological substrate of SHP2, the EGFR, was
identified and characterized. Subsequently, a molecular mechanism
for SHP2 in promoting EGFR signaling was described. It was
demonstrated that SHP2 promotes Ras activation by interfering with
the process of Ras inactivation catalyzed by the Ras GTPase
activating protein (RasGAP). Inhibition is achieved through the
dephosphorylation of Tyr992 of the EGFR, which serves as RasGAP
binding site (5).
[0015] Substrate-trapping and mass spectroscopic analysis showed
that a-catenin also is a biological substrate of SHP2 (23). Tyr
phosphorylation of .alpha.-catenin enhances its translocation to
the plasma membrane and its interaction with .beta.-catenin,
leading to enhanced actin polymerization and stabilization of
adherens junction-mediated intercellular adhesion, a phenomenon
commensurate with loss of the transformation phenotype.
Dephosphorylation of .alpha.-catenin by SHP2 suppresses
intercellular adhesion and increases the cytosolic pool of
.beta.-catenin and its subsequent translocation to the nucleus (23)
where it acts as a transcription factor for mitogenic genes such as
cyclin D1 and c-myc (1, 13). In addition, inhibition of
.alpha.-catenin enhances activation of the Ras-ERK and the PI3K-Akt
pathways (23, 97), induces hyperproliferation of skin epithelium
(97) and promotes cell transformation (22, 63). Therefore, SHP2
mediates .beta.-catenin activation downstream of RTKs bypassing the
need for Wnt ligand stimulation (23). The previously held notion
was that .beta.-catenin is activated downstream of the frizzled
(FZ) and low-density lipoprotein related protein ("LRP.sup.5/6")
coreceptors following Wnt ligand stimulation (18, 49, 60).
[0016] In EGFR and HER2, SHP2 dephosphorylates negative-regulatory
phosphorylation sites that serve as RasGAP binding sites. By doing
so, SHP2 promotes the Ras-ERK and the PI3K-Akt signaling pathways
(5). In .alpha.-catenin, SHP2 dephosphorylates a phosphotyrosyl
that mediates interaction with .beta.-catenin (23). This leads to
suppression of adherens junction-mediated cell-cell interaction, an
increase in cytosolic .beta.-catenin pool and subsequent
translocation of .beta.-catenin to the nucleus where it acts as a
transcription factor for mitogenic genes. Thus, through SHP2, EGFR
and HER2 can activate, not only the Ras-ERK and the PI3K-Akt
pathways, but also .beta.-catenin signaling. Therefore, mediation
of cell transformation by SHP2 is a complex process that involves
modulation of the Ras-ERK and PI3K-Akt signaling pathways,
intercellular adhesion, focal adhesion and actin cytoskeletal
reorganization. These findings suggest that SHP2 could be a
potential target for cancer treatment.
[0017] Examples that highlight the importance of SHP2 in growth
factor signaling, cell transformation and cancer development are
provided below. These results were based on inhibition of SHP2
either by dominant-negative SHP2 (DN-SHP2) expression or small
interfering RNA (Si-RNA)-mediated ablation of the SHP2 protein. As
known in the art, DN-SHP2 expression refers to ectopic expression
of a dysfunctional mutant protein that competes with the endogenous
counterpart and inhibits function. The controls for these
experiments were vector alone and the wild type form of SHP2
(WT-SHP2). The indicated breast cancer cell lines were infected
with retrovirus expressing vector alone, WT-SHP2 or DN-SHP2, and
stable lines from each group were seeded in soft agar, a commonly
used assay for testing anchorage-independent growth (3, 23). The
MCF10A, the immortalized normal breast cell line, was used as a
negative control for anchorage-independent growth. Expression of
DN-SHP2 inhibited colony formation by all of the breast cancer
cells used in this experiment, while expression of vector alone or
WT-SHP2 did not cause any change (FIG. 1), As expected, the
negative control MCF10A cells could not form colonies in soft agar
during anchorage-independent growth studies. In addition,
expression of DN-SHP2 modestly suppressed cell growth in all of the
cells used in this study (FIG. 2). Together, these results show
that SHP2 is important for cell proliferation and
anchorage-independent growth of breast cancer cells.
[0018] The importance of SHP2 in cell transformation and growth
factor signaling was further investigated by Si-RNA-mediated
ablation of the SHP2 protein in the BT474 breast cancer cells that
overexpress the HER2 oncogene. As known in the art, expression of
Si-RNA in cells inhibits the translation of the corresponding
messenger RNA (mRNA) by hybridizing to a specific complementary
region within the mRNA. For the current work, stable cell lines
harboring the SHP2 Si-RNA were produced by infection of the BT474
cells with retroviruses expressing the SHP2 Si-RNA under the
control of a tetracycline-inducible system (BD Biosceinces). These
cells express the SHP2 Si-RNA only when they were treated with
tetracycline. Parent BT474 cells were used as controls (control) in
these experiments. In addition, cells harboring the SHP2 Si-RNA
(non-Si-RNA), but not treated with tetracycline were used as
controls for the effect of retroviral infection and gene
integration. Therefore, the experimental groups used in these
studies included controls, non-Si-RNA and Si-RNA cells.
Si-RNA-mediated ablation of the SHP2 protein induced reversion of
the BT474 cells to a normal phenotype that compares with the
cobblestone-like appearance of the MCF10A, the immortalized normal
breast epithelial cell line (FIG. 3). It also inhibited anchorage
independent growth in soft agar (FIG. 4) and led to
re-differentiation of the BT474 cells back to normal as evidenced
by acini formation in laminin-rich basement membrane (LRBM)
cultures (FIG. 5). It should be noted that Si-RNA cells die in soft
agar, but re-differentiate to normal under adherent two dimensional
and LRBM cultures. These results demonstrate that SHP2 is important
for the maintenance of the transformation phenotype of breast
cancer cells. In addition, they show that interference with SHP2
function leads to re-differentiation of breast cancer cells to a
normal phenotype.
[0019] In addition to morphological and growth behavior changes,
Si-RNA-mediated ablation of the SHP2 protein inhibited EGF-induced
signaling. The indicated cells were grown to subconfluency, serum
starved for about 12 hours and then stimulated with 10 ng/ml EGF
for varying time points. Lysates prepared from these cells were
separated by denaturing polyacrylamide gel electrophoresis,
transferred onto a nitrocellulose membrane and analyzed by western
blotting with antibodies that recognize the activated forms of
ERK1/2 and Akt. Si-RNA-mediated ablation of the SHP2 protein
inhibited EGF-induced activation of these proteins (FIG. 6),
suggesting that SHP2 is required for mitogenic and cell survival
signals in breast cancer cells. Therefore, SHP2 has the potential
to serve as a new drug target for the treatment of breast
cancer.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0020] FIG. 1 is a colony formation in soft agar by several breast
cancer cells expressing vector alone, WT-SHP2 and DN SHP2.
[0021] FIG. 2 is suppression of cell proliferation by the
expression of DN-SHP2 in the BT474 breast cancer cell line.
[0022] FIG. 3 is induction of a normal epithelial phenotype in the
BT474 breast cancer cells by Si-RNA-mediated ablation of the SHP2
protein
[0023] FIG. 4 is inhibition of anchorage-independent growth ability
of the BT474 breast cancer cells in soft agar by Si-RNA-mediated
ablation of the SHP2 protein.
[0024] FIG. 5 is induction of re-differentiation in the BT474
breast cancer cells to a normal acin-forming breast epithelial
cells.
[0025] FIG. 6 is inhibition of epidermal growth factor-induced
activation of ERK1/2 and Akt in the BT474 breast cancer cells by
Si-RNA-mediated ablation of the SHP2 protein.
[0026] FIG. 7 are the functional groups for the modification of the
WGMDY peptides at the Tyr residue or the R' modification of the
SEBA derivatives.
[0027] FIG. 8 is 4-(2-sulfaminoethyl)benzoic acid (SEBA).
[0028] FIG. 9 are the SEBA derivatives
[0029] FIG. 10 are the oligopeptides for modification at the R
position of SEBA derivatives.
[0030] FIG. 11 is the anti-SHP2 peptide pWGMDY1 inhibition of
phosphatase activity of SHP2.
[0031] FIG. 12 is the SEBA inhibition of phophastase activity of
SHP2 shown in a protein gel.
[0032] FIG. 13 is the effect of pWGMDY on EGF-induced ERK1/2 and
AKT activation.
BRIEF SUMMARY OF THE INVENTION
[0033] The present invention pertains to the inhibition of SHP2 in
vivo and in vitro. The mechanism of inhibition is the binding of
specific inhibitors to the active of SHP2.
[0034] The present invention further details peptides that can
inhibit the function of SHP2. The anti-SHP2 peptides are
oligopeptides that are 3-5 amino acids long, in their native form
or their conservative variants. These peptides have been given a
common name termed WGMDY
[0035] Another aspect of the present invention is the ability to
modify the anti-SHP2 peptides to create conservative variants. In
this application the modification to create a conservative variant
means: a) replacement of one amino acid by another to create a
conservative variant; b) addition of a phosphate, a sulfate,
amino-sulfate or an acetyl group to the single Tyr residue, c)
introducing changes to side chains of amino acids, and d) making
changes to the peptide backbone of the peptides.
[0036] Another object of the present invention is the chemical
compound 4-(2-sulfaminoethyl) benzoic acid, SEBA (FIG. 8), and the
use of this compound with anti-SHP2 function.
[0037] Another aspect of the present invention is the use of SEBA
derivatives in the inhibition of the SHP2 function. The SEBA
derivatives have the chemical formula
##STR00001##
wherein R is selected from the functional group consisting of the
amino acids sequence D-V; D-G; D-A; D-G-D-G; D-A-D-A; D-V-D-V;
D-A-D-V; D-A-D-G; D-G-D-V; D-G-D-A; D-V-D-G; and D-V-D-A (also see
FIG. 10) and R' is selected from the functional groups acetate;
phosphate, sulfate, and aminosulfate (also see FIG. 7).
[0038] A further aspect of the invention is the inhibition of SHP2
in the treatment of human disease, namely cancer.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention involves 1) anti-SHP2 peptides and
their conservative variants and 2) a peptidomimetic anti-SHP2
compound SEBA and its derivatives for use in inhibiting SHP2
function both in vitro and in vivo. It also teaches that SHP2 is a
potential therapeutic target for the treatment of cancer and that
interfering with SHP2 function using the anti-SHP2 peptides and
SEBA compounds reverses cell transformation and induces remission
of preformed tumors in vivo. As used herein, the term anti-SHP2
peptides refers to oligopeptides that are 3-5 amino acids long, in
their native form or their conservative variants. These peptides
have been given a common name termed WGMDY. Conservative variants
of WGMDY peptides means: a) replacement of one amino acid by
another to create a conservative variant; b) addition of a
phosphate, a sulfate, amino-sulfate or an acetyl group to the
single Tyr residue, c) introducing changes to side chains of amino
acids, and d) making changes to the peptide backbone of the
peptides. Detailed sequences of WGMDY peptides (FIG. 7) and
functional groups for modification at the Y (Tyr) residue (FIG. 8)
are provided.
WGMDY Peptides
TABLE-US-00002 [0040] SEQ ID: 1 LENGTH: 5 SEQUENCE:
Asp-Ala-Asp-Val-Tyr SEQ ID: 2 LENGTH: 5 SEQUENCE:
Asp-Ala-Asp-Gly-Tyr SEQ ID: 3 LENGTH: 5 SEQUENCE:
Glu-Ala-Asp-Val-Tyr SEQ ID: 4 LENGTH: 3 SEQUENCE: Asp-Val-Tyr SEQ
ID: 5 LENGTH: 3 SEQUENCE: Asp-Gly-Tyr
[0041] Conservative amino acid substitutions within the context of
the application are shown in the table below and can be used to
create conservative variants which have changes in the amino acid
sequence, but are similar in function due to the conservative
nature of the change. A conservative change is best described as
the substitution of an acidic amino acid for an acidic amino acid
or a hydrophobic amino acid for a hydrophobic amino acid. The
exchange of an amino acid for another amino acid of similar
chemical properties creates a conservative variant.
TABLE-US-00003 Amino Acid Conservative variants Aspartic Acid, Asp,
D Glutamic Acid, Glu, E Glycine, Gly, G Alanine, Ala or A Alanine,
Ala, A Glycine, Gly, G Valine, Val, V Leucine, Leu, L Isoleucine,
Ile, I Valine, Val, V Glycine, Gly, G Alanine, Ala, A Leucine, Leu,
L Isoleucine, Ile, I Tyrosine, Tyr, Y Phenylalanine, Phe, F
[0042] As also used herein, the term SEBA refers to
4-(2-sulfaminoethyl)benzoic acid (FIG. 8), while the term SEBA
derivatives refers to modified version of SEBA derivative shown in
FIG. 9. Modifications to produce SEBA derivatives include covalent
linkage of functional groups of FIG. 7 to the R' and amino acid
oligomers or their modified counterparts of FIG. 11 to the R.
Modifications to amino acid oligomers linked to the R position
refer to changes to the peptide bonds so as to confer resistance to
peptidases or proteases, addition of hydrophobic groups or
penetrating peptide sequences to enhance cellular uptake and
changes to side chains of constituent amino acids to increase
binding affinity to the target. In this context, the target is the
active site of the SHP2 phosphatase domain.
[0043] In the context of the present invention, inhibition of SHP2
with WGMDY peptides and SEBA compounds refers to interfering with
the normal function of SHP2. Inhibition is achieved by physical
interaction of the WGMDY peptides and SEBA compounds with the
active site of the phosphotyrosyl phosphatase domain of SHP2. This
interaction blocks the binding of biological substrates to the same
site. The overall effect is an increase in the Tyr phosphorylation
level of biological substrates, leading to abrogation of the signal
transducer role of SHP2. As described in the background and
supporting evidences, the major signaling networks that are
affected by SHP2 inhibition are the Ras-ERK, the PI3K-Akt and
.beta.-catenin signaling pathways. Consequently, promotion of cell
proliferation, survival, transformation, migration and tumor growth
by SHP2 is inhibited. The outstanding findings presented in FIGS.
1-6 clearly show that inhibition of SHP2 has the potential for the
treatment of cancer. In addition, recent reports by the inventor
(5, 23, 61) lend further support to this invention.
[0044] As outlined above, biological substrates of SHP2 were
isolated by substrate-trapping techniques and then identified by a
combination of mass spectroscopic and immunoblotting analysis (4,
5, 23). By employing site-directed mutagenesis, binding studies and
functional analysis of mutants, target Tyr residues for the SHP2
phosphatase activity were identified (23, 61). Further
site-directed mutagenesis and binding studies and computer-based
comparison (FASTA) of amino acid sequences surrounding target Tyr
residues showed a potential consensus motif for recognition of
substrates by the active site of the SHP2 phosphatase domain. The
characteristic features of this consensus motif are the Y (Tyr)
residue that acts as a substrate when phosphorylated, the
invariable D (Asp) residue at the -2 position (the amino acid
immediately N-terminal to the Y is referred to as -1), hydrophobic
residues at the -1 and -3 positions and a D or E (Glu) residue at
the -4 position (FIG. 7). As is known in the art, amino acids in
WGMDY peptides are covalently linked to each other through what is
known as peptide bond. As is also known in the art, peptide bonds
are formed between the carboxyl group of the N-terminal amino acid
and the amine group of the next amino acid and so on.
[0045] Accumulating evidence indicates that SHP2 could serve as a
therapeutic target for the treatment of cancer. In the context of
the current invention, this conclusion is based on the following
findings. First, SHP2 is a positive effector of EGFR and HER2
signaling and cell transformation receptors. And secondly, SHP2 is
required for EGFR- and HER2-induced activation of 13-catenin, the
major transducer of the Wnt signaling pathway. Thus, SHP2
integrates the EGFR/HER2 and 13-catenin signaling pathways. Because
aberrant signaling by the EGFR, HER2 and .beta.-catenin is
implicated in the development of a variety of cancers, inhibition
of SHP2 has the potential for cancer treatment
[0046] Aberrant EGFR and HER2 signaling is strongly associated with
aggressive tumor growth which correlates with poor prognosis for
patient survival. As a result, there has been a great deal of
interest in the production of drugs that inactivate EGFR and HER2
in tumors. Approximately 50% of breast, ovarian, colorectal and
lung cancers overexpress EGFR or HER2 due to gene amplification,
and of these, the majority have gene rearrangements that result in
constitutive activation of these receptors (44, 64, 67, 77, 101).
Currently, several drugs that interfere with the functions of these
proteins have been produced. Some are already in clinical use while
others are in advanced stage of clinical trials. However, there
have not been any breakthroughs. For instance, herceptin, the
anti-HER2 drug currently in clinical use for breast cancer, slows
tumor progression in only 30% of the patients. In the pipeline are
drugs that interfere with the Tyr kinase domains of EGFR and HER2,
most notably, traceva (erlotinib) and iressa (gefitinib). Traceva
and iressa are currently in advanced clinical trials.
Unfortunately, the outcome of these trials has not been promising.
For instance, patients treated with standard chemotherapy in
combination with Iressa did not show any significant increase in
overall survival rates than those treated with chemotherapy alone
(31, 68, 75). Although these drugs provide some benefit by
lengthening the life of the patient by several months to few a
years, the cancer usually recurs and the patient eventually dies
even after combination therapy with other non-specific agents such
as paclitaxel and cisplatin. The common problem in the treatment of
breast and other forms of cancer has been the ability of a cancer
cell to adjust and overcome targeted chemotherapy by activating
parallel growth signals. Because SHP2 is an essential downstream
effector of EGFR and HER2, inhibition of SHP2 with drugs that will
be developed from pWGMDY and SEBA derivatives will have a much
better outcome than anti-EGFR and anti-HER2 drugs.
[0047] Increased Wnt signaling either due to the presence of an
abnormally high amount of Wnt ligands or defects in one or more of
the multiprotein complexes that channel cytoplasmic .beta.-catenin
for degradation, results in stabilization of .beta.-catenin leading
to its translocation to the nucleus where it induces the expression
of mitogenic genes (11, 16, 18, 20, 37, 60). In support of this
phenomenon, MMTV-LTR-driven overexpressing of Wnt1 or
constitutively active .beta.-catenin or knockout of a-catenin
expression consistently resulted in a hyperplastic breast tissue
(40, 91). Therefore, biochemical defects that ultimately result in
deregulated .beta.-catenin can induce epithelial to mesenchymal
transition ("EMT") leading to cell transformation and tumor growth
in the breast or other tissues. The similarity of a-catenin
knockout to constitutively active .beta.-catenin indicate that
interference with a-catenin could also result in .beta.-catenin
stabilization and translocation to the nucleus. The findings in
fibroblasts transformed with the constitutively active FGFR3 (23)
and in the BT474 breast cancer cell line showed that SHP2
interferes with Tyr phosphorylation-dependent interaction of
a-catenin with .beta.-catenin. Therefore, under conditions of
elevated Tyr phosphorylation, such as overexpression of EGFR or
HER2, SHP2 can activate .beta.-catenin even without Wnt
stimulation.
[0048] To provide evidence on the use of WGMDY peptides to inhibit
SHP2 function, WGMDY1 was fused to a 10-mer cell-penetrating
peptide sequence of HIV tat-1 protein (85, 90, 94, 98) to aid
internalization and custom synthesized as a phosphorylated form
(pWGMDY1) and unphosphorylated form (WGMDY1). The anti-SHP2 effect
of these preparations were first tested by in vitro phosphatase
assays using purified glutathione S-transferase (GST) fusion of the
phosphatase domain of SHP2 (denoted as P) as an enzyme and
para-nitrophenolphosphate (denoted as pN) as an artificial
substrate as described recently (61). Reactions containing buffer,
P and pN only were used as negative controls for the
dephosphorylation reactions. Para-nitrophenol phosphate (pN) is
colorless in solution and as such has very low optical density.
But, when dephosphorylated to para-nitrophenol, it exhibits a
yellowish discoloration, acquiring an increased optical density
that can be measured with a spectrophotometer. Absorption of
visible light by para-nitrophenol phosphate peaks at a wave length
of 405 nm. Therefore, reactions were followed with absorbance
measurement (OD) at 405 nm in a plate reader spectrophotometer
(TECAN). Absorbance at the 5 minutes time point was used for
comparing the effect of pWGMDY1 on the phosphatase activity of P.
As described previously, dephosphorylation of pN by P was more than
15-fold over basal after a 5 minute incubation period (61).
Addition of pWGMDY1, but not WGMDY1, inhibited the
dephosphorylation reaction (FIG. 11). Similar results were obtained
when SEBA was used in place of pWGMDY (FIG. 12). These results
demonstrate that pWGMDY1 and SEBA are SHP2 inhibitors.
[0049] To determine that pWGMDY1 can also be used to inhibit SHP2
in cells, the following experiments were performed. The BT474
breast cancer cells were treated with 5 .mu.g/ml of pWGMDY1 or
WGMDY1 for 30 minutes. They were then stimulated with EGF for
varying time points. Total cell lysates prepared from these cells
were analyzed by immunoblotting with specific antibodies that
recognize the activated forms of these proteins. As shown in FIG.
13, pWGMDY inhibited ERK1/2 and Akt activation, while WGMDY did
not. Note that ERK1/2 and Akt activation was sustained for more
than 2 hours in the control WGMDY1-treated cells, while it was
submaximal even at the 10 minute time point in the pWGMDY 1-treated
cells. Anti-B-actin reblotting showed that total protein levels
were comparable. These results demonstrate that pWGMDY1 inhibits
the signal transduction role of SHP2 in intact cells.
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Sequence CWU 1
1
515PRTHomo sapiens 1Asp Ala Asp Val Tyr1 525PRTHomo sapiens 2Asp
Ala Asp Gly Tyr1 535PRTHomo sapiens 3Glu Ala Asp Val Tyr1
543PRTHomo sapiens 4Asp Val Tyr153PRTHomo sapiens 5Asp Gly Tyr1
* * * * *