pubs.acs.org/Biochemistry Published on Web 07/11/2010
r2010 American Chemical Society
Biochemistry 2010, 49, 6687–6695
Structure-Activity Relationship Analysis of Peptides Targeting the EphA2 Receptor†
Srinivas Duggineni,‡,),^Mitchell Koolpe, Xuejun Zhu,‡Ziwei Huang,‡,§,^and Elena B. Pasquale*,‡,§
‡Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, and
§Department of Pathology, University of California, San Diego, California 92093
to this work.
State University of New York, Syracuse, NY 13210.
These two authors contributed equally
^Current address: Department of Pharmacology, SUNY Upstate Cancer Research Institute,
Received April 22, 2010; Revised Manuscript Received June 17, 2010
ABSTRACT: The EphA2 receptor tyrosine kinase has emerged as a promising new therapeutic target in cancer
because of its high level of expression in tumors. EphA2-specific antibodies have been used to deliver drugs
and toxins to tumor cells, leading to inhibition of tumor growth and metastatic dissemination. We previously
identified two related peptides, YSA and SWL, that selectively bind to the ligand-binding domain of EphA2
but not other Eph receptors and could therefore be useful as selective targeting agents. Here we characterize
residues appear to be critical for high-affinity receptor binding. Furthermore, a peptide comprising only the
first five residues of YSA retains selectivity for EphA2. Similar to ephrin-A1, the physiological ligand for
EphA2, both YSA and SWL activate EphA2 and inhibit downstream oncogenic signaling pathways in PC3
cancer cells. The two peptides and derivatives are quite stable in conditioned cell culture medium and show
promise for delivering drugs and imaging agents to EphA2-expressing tumors.
The EphA2 receptor tyrosine kinase, a member of the large
Eph receptor family, is a promising therapeutic target in cancer
breast, ovarian, prostate, pancreatic, and lung cancer (1-3).
vasculature, while it is undetectable in normal quiescent vascu-
lature (4, 5). Furthermore, high EphA2 levels have been asso-
ciated with a poor clinical prognosis (1-3) and with the more
malignant, basal type of breast and prostate cancers (6, 7).
EphA2 overexpression has indeed been shown to induce onco-
genic transformation and invasiveness of cultured mammary
epithelial cells (8), and EphA2 downregulation with siRNA or
antisense oligonucleotides has a negative impact on tumor
growth and metastasis in mouse cancer models (9, 10). Interest-
ingly, EphA2 is tyrosine phosphorylated (activated) at low to
undetectable levels in most tumors, suggesting an oncogenic role
that is independent of ligand-mediated activation (11-13).
Five glycosylphosphatidylinositol-linked ligands (ephrin-A1
to ephrin-A5) can induce EphA2 tyrosine phosphorylation and
that ephrin-induced EphA2 activation inhibits major oncogenic
signaling pathways, such as the Ras-MAP kinase pathway and the
PI3 kinase/Akt pathway, as well as celltransformation (11, 13, 15).
Therefore, EphA2 functions as a tumor suppressor when its
signaling ability is activated by ephrin ligands, whereas its tumor
promoting effects may be ligand-independent (3, 12, 13, 16).
A number of EphA2-targeting agents have been developed.
Several agonistic monoclonal antibodies and ephrin-A1 Fc, a
solubleformof ephrin-A1,havebeenshowntodecreasethe level
oftumorgrowth and metastasis inmousemodels (17-22).Their
downstream signaling pathways with tumor suppressor activity,
(2) receptor internalization and degradation, possibly accompa-
nied by presentation of EphA2-derived peptides recognized by
effector T cells, and (3) antibody-dependent immune cell-medi-
ated cytotoxicity. A bispecific antibody engineered to simulta-
nously bind EphA2 and the T cell receptor-CD3 complex has
also been shown to effectively promote destruction of EphA2-
expressing tumor cells (23). Furthermore, gold-coated silica
nanoshells conjugated to ephrin-A1 have been used for targeted
photothermal ablation of cultured PC3 prostate cancer cells,
which express high levels of EphA2 (24).
Ephrin ligands and agonistic antibodies that cause EphA2
internalization can also be used to deliver drugs or toxins to
tumors. Ephrin-A1 conjugated to Pseudomonas aeruginosa exo-
toxin A has been shown to kill EphA2-expressing cancer cells in
culture(25).Furthermore,an EphA2-specificantibody conjugated
to a derivative of auristatin, a drug that disrupts microtubules,
dramatically inhibits tumor growth in animal models (26, 27).
Finally, EphA2 antibodies coupled to imaging agents have been
successfully used for tumor visualization in mouse xenograft
models (28). This could be useful for cancer diagnosis, particularly
because EphA2 appears to be overexpressed starting from early
stages of cancer (4).
As an alternative to the use of ephrins or antibodies, we have
identified two related EphA2-targeting peptides by using phage
display (29). These 12mer peptides, designated YSA and SWL,
selectively bind to the ephrin-binding domain of EphA2 but not
other Eph receptors and thus may be used as selective targeting
agents. Both peptides also inhibit ephrin binding to EphA2.
†This work was supported by National Institutes of Health Grant
CA82713 (to E.B.P.), a grant from MedImmune (to E.B.P.), Depart-
1-0462 (to Z.H. and E.B.P.), Prostate Cancer Research Program Grant
W81XWH-06-1-0077 (to E.B.P.),
DAAMD17-01-1-0168 (to M.K.).
*To whom correspondence should be addressed: Sanford-Burnham
Medical Research Institute, 10901 N. Torrey Pines Rd., La Jolla, CA
92037. Phone: (858) 646-3131. Fax: (858) 646-3199. E-mail: elenap@
6688 Biochemistry, Vol. 49, No. 31, 2010Mitra et al.
In addition, the YSA peptide, which has been more extensively
characterized, was shown to induce EphA2 tyrosine phosphor-
ylation and inhibition of Erk MAP kinases1in endothelial
cells (29). Therefore, the YSA peptide is an agonist capable of
activating EphA2 signaling. We also previously showed that the
YSA peptide, fused to the pIII coat protein of M13 phage, can
Other groups have demonstrated the usefulness of YSA, con-
jugated to magnetic nanoparticles, for targeting and removal of
cancer cells from the ascites fluids of mice and ovarian cancer
delivery of siRNA-loaded nanogels used to chemosensitize
cancer cells expressing EphA2 (32, 33). In addition, adenoviral
vectors engineered to include the YSA peptide in the HI loop of
their fiber cell-binding protein have been successfully targeted to
EphA2-expressing pancreatic cancer cells in vitro (34). In vivo
targeting of EphA2-expressing cells with the engineered adeno-
virus, however, was not successful, presumably because of
insufficient binding affinity of the peptide inserted into the
adenoviral protein (34).
peptide identified by phage display. However, structure-activity
analysis of the YSA peptide, and also the SWL peptide, could
provide useful information for further optimization to generate
more effective EphA2-targeting agents. Here we identify the
residues of the YSA peptide that are critical for EphA2 binding
and characterize additional derivatives of the YSA and SWL
peptides. We also demonstrate that the YSA and SWL peptides
both peptides can activate the EphA2 receptor and inhibit
phosphorylation of Akt and Erk MAP kinases in PC3 prostate
cancer cells. These peptides, and derivatives with improved
binding affinity, represent promising candidates as EphA2-
targeting agents in cancer.
Peptide Synthesis. Some peptides were purchased from
Neobioscience (Boston, MA), and others were synthesized auto-
matically on an Applied Biosystems 433A (Applied Biosystems,
Foster City, CA) peptide synthesizer by using Fmoc [N-(9-
fluorenyl)methoxycarbonyl] chemistry. Low loading Tenta Gel S
RAM amide resin (loading, 0.24 mmol/g; Fluka) was used for the
in-house synthesis. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate (HBTU) and N-hydroxybenzo-
triazole (HOBt) in N,N-dimethylformamide (DMF) were used as
coupling and activating reagents in the presence of diisopropy-
lethylamine (DIEA). Fmoc group deprotection at each step was
conducted using piperidine in NMP (20%). Side chain deprotec-
tion and cleavage were performed by treatment with a mixture of
trifluoroacetic acid (TFA), H2O, thioanisole, phenol, and ethane-
dithiol (82.5:5:5:5:2.5, v/v/v/v, 5 mL) at room temperature for
2.5 h. TFA was removed by evaporation, and the crude peptides
were precipitated with cooled tert-butylmethyl ether, washed
were characterized by matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) mass spectrometry. The purity of
the crude peptides was 82-94% as measured by HPLC, and
HPLC traces revealed in each case only one major peak and
some minor impurities. The peptides used for the experiments in
tail and purified as previously described (29).
The crude YSA-C-10mer peptide was dissolved in an 80:20
CH3CN/H2O mixture and purified using semipreparative reverse-
phase high-performance liquid chromatography (RP-HPLC).
Fractions containing the peptide were pooled together and lyoph-
ilized. Dimerization of the purified peptide was performed in 1 M
of peptide/mL of buffer) and was monitored by analytical RP-
HPLC using a Vydac C-18 column [5 μm, 150 mm (length) ? 4.6
solvents were used: A, water with 0.1% TFA; B, 20% water in
CH3CN with 0.1% TFA in a linear 10 to 65% gradient over
30 min. The purity of the dimerized YSA-C-10mer was 99%. For
DTT treatment of the YSA-C-10mer peptide (containing an un-
2 mM DTT (20 μL total volume) for 20 min at room temperature,
to ensure that no intermolecular disulfide bonds were formed. The
1 mM stock was then diluted to 0.5-50 μM for the ELISA.
ELISAs. To assess the binding of EphA2 Fc to immobilized
peptides, streptavidin-coated wells were incubated with 1 μM
biotinylated peptides for 1 h at room temperature, rinsed with
Tris buffer [150 mM NaCl and 50 mM Tris-HCl (pH 7.5)]
indicated concentrations of EphA2 Fc (R&D Systems, Minnea-
polis, MN) for 1 h at room temperature. After the unbound
EphA2 Fc had been washed away with the same buffer, the
plate was incubated with an anti-Fc antibody conjugated to AP
(anti-Fc AP) (Promega, Madison, WI) for 1 h to measure the
amount of bound EphA2 Fc. Bound anti-Fc AP was detected
using 5.4 mM p-nitrophenol phosphate (pNPP) substrate in
SEAP buffer [0.22 M diethanolamine and 1 mM MgCl2(pH
9.8)] and measuring the absorbance at an OD of 405 nm.
To determine the Eph receptor selectivity of the YSA-5mer
peptide, streptavidin-coated wells were incubated with 5 μM
biotinylated peptide for 1 h at room temperature, followed by
Systems) for 1 h. Bound Eph Fc receptors were then detected
using the anti-Fc AP antibody as described above.
FIGURE 1: (A) Alignment of the sequences of the YSA and SWL
two tyrosines in the YSA peptide are critical for binding to EphA2.
The curves show the binding of EphA2 Fc to immobilized biotiny-
lated YSA peptide or peptides in which Y1 or Y4 is mutated to
alanine. Similar amounts of the three peptides were immobilized on
streptavidin-coated ELISA wells.
1Abbreviations: CD3, cluster of differentiation 3; DAPI, 40,6-diami-
dino-2-phenylindole; DTT, dithiothreitol; ELISA, enzyme-linked im-
munosorbent assay;GPI, glycosylphosphatidylinositol; HUVE, human
umbilical vein endothelial; MAP kinase, mitogen-activated protein
kinase; PDB, Protein Data Bank; PI3 kinase, phosphatidylinositol
Article Biochemistry, Vol. 49, No. 31, 20106689
To measure the IC50values of the peptides, protein A-coated
wells (Pierce Biotechnology, Rockford, IL) were incubated with a
and 50 mM Tris-HCl (pH 7.5)] containing 0.02 mM Tween 20 for
1 h. EphA2 Fc-coated wells were then rinsed with Tris buffer and
0.01% Tween 20 and incubated with different peptide concentra-
tions and 0.6 nM ephrin-A5 AP (0.94 OD min-1mL-1) in a total
pNPP substrate. Data were fitted using nonlinear regression, and
Molecular Docking. Computer docking of the YSA peptide
to the EphA2 ligand-binding domain (PDB entry 3HEI) was
performed using the ZDOCK module of DiscoveryStudio 2.1
(Accelrys, Inc., San Diego, CA). Residues defined as being impor-
tant for the interaction of the peptide with the ephrin-binding
score, high density, and low cluster number were typed with the
CHARMm Polar H force field and refined with RDOCK. The
docking pose with the lowest RDOCK energy was chosen for
detailed analysis and display.
Determination of Peptide Stability. For the determination
peptidesinPC3 conditionedmedium was incubated for5 daysin
a 37 ?C incubator. The peptides were then used in the ELISA
described above for determination of IC50values, at concentra-
tions calculated on the basis of the original concentration before
Measurements of EphA2 Activation and Downstream
Signaling. PC3 prostate cancer cells were grown in RPMI 1640
medium(Mediatech,Inc.,Herndon,VA) supplementedwith 10%
fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1%
sodium pyruvate and then serum-starved overnight in medium
without FBS. For measurements of EphA2 tyrosine phosphoryla-
tion, the cells were then treated for 20 min with 0.1 μg/mL human
Fc (as a negative control), 0.1 μg/mL ephrin-A1 Fc (as a positive
control), or 50 μM peptides. The cells were then rinsed once with
ice-cold PBS, lysed in modified RIPA lysis buffer [150 mM NaCl,
1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
1 mM sodium orthovanadate, and centrifuged at 16000g for
10 min. For immunoprecipitations, 25 μL of GammaBind G
Sepharose beads (GE Health Care Life Sciences, Piscataway,
NJ) were rinsed twice with modified RIPA buffer and incubated
for 3 h at 4 ?C with 3 μg of anti-EphA2 antibody (Millipore-
Upstate, Inc., Temecula, CA) and the cell lysates. The immuno-
precipitates were resolved by gel electrophoresis on 4 to 20% Tris-
glycine gels (Invitrogen, Carlsbad, CA) and transferred to Im-
mobilon-P transfer membranes (Millipore Corp., Bedford, MA),
which were subsequently probed with horseradish peroxidase
(HRP)-conjugated anti-phosphotyrosine antibody (PY20; BD
Transduction Laboratories, Inc.), and reprobed with an anti-
EphA2 antibody (Zymed/Invitrogen, Carlsbad, CA).
To measure the effects of the peptides on Akt and Erk1/Erk2
phosphorylation, PC3 cells were serum-starved overnight, pre-
treated with 50 μM peptides for 10 min, and then treated with
10% FBS in the continued presence of the peptides for 10 min.
Controls in the absence of the peptides included cells stimulated
with 1 μg/mL ephrin-A1 Fc along with FBS for 10 min and cells
not stimulated with FBS. Cells were then lysed in RIPA lysis
buffer as described above, and proteins were resolved by gel
electrophoresis and probed by immunoblotting as described
above using antibodies to phospho-Akt (Thr308), total Akt,
phospho-p44/42 MAPK (Erk1/2), and total Erk (Cell Signaling
Technology, Inc., Danvers, MA).
Cell Retraction Assay. PC3 cells were grown for 48 h on
glass coverslips and serum-starved for 4 h. The cells were then
treated with 100 μM YSA-12mer or SWL-12mer for 30 min or
coverslipswere then washed with cold PBS, fixed in 4% formalde-
hyde and 4% sucrose for 10 min, and permeabilized with 0.5%
Triton X-100 for 3 min. The coverslips were then washed with 1?
PBS, blocked in 2% normal goat serum (NGS) and 2% BSA in
PBS for 1 h at room temperature, and incubated with rhodamine-
conjugated phalloidin (diluted 1:300 in PBS containing 2% non-
immune goat serum) for 1 h at room temperature. After being
washed with PBS, the coverslips were mounted on microscope
slides with Vectashield mounting medium containing DAPI
(Vector Laboratories, Burlingame, CA). Images were collected
using an Inverted TE300 Nikon wide-field fluorescence micro-
scope and processed using Adobe Photoshop.
RESULTS AND DISCUSSION
Two Tyrosines in the YSA Peptide Are Critical for EphA2
motif [where X represents any amino acid (Figure 1A)]. Because
tyrosine and tryptophan are known to be critical residues for
mediating molecular contacts in protein-protein interfaces (36),
we examined the importance of the two tyrosine residues in the
first tyrosine (Y1) resulted in a marked decrease in the level of
bindingofthe EphA2ligand-binding domainfused tohuman Fc
(EphA2 Fc) to the immobilized peptide in ELISAs (Figure 1B).
Binding of EphA2 Fc to the YSA peptide was no longer detec-
table when the second tyrosine (Y4) was changed to alanine
(Figure 1B). These results illustratethe critical importance of the
two tyrosine residues at positions 1 and 4 of the YSA peptide.
Only 5 of the 12 Residues in the YSA Peptide Are
available about the pharmacophoric elements involved in the
binding of the YSA peptide to the EphA2 receptor, we utilized a
systematic alanine scanning approach to identify residues impor-
tant for potency. Eleven YSA peptide derivatives were synthesized
with alanine at each position (Figure 2A). The YSA peptide was
denotedasAla-3because it alreadyhasanalanineat that position.
Fc immobilized on protein A-coated plates was incubated with
ephrin-A5 alkaline phosphatase (AP) ligand in the presence of
varying peptide concentrations, and the amount of ephrin-A5 AP
bound to EphA2 was determined. The results confirm the im-
portance of Y1 and Y4, because the Ala-1 and Ala-4 peptides lost
the ability to inhibit ephrin-A5 AP binding to EphA2. The Ala-5
peptidealso didnotdetectablyinhibit EphA2-ephrin-A5binding,
indicating that P5 is also critical for the high binding affinity of the
YSA peptide for EphA2 (Figure 2). This suggests that the proline,
which is also present in the SWL peptide (Figure 1A), may help
bend the peptide to fit in the EphA2 ephrin-binding channel.
binding, because the Ala-6 peptide had barely measurable inhibi-
tory activity. The Ala-8 and Ala-9 peptides had a 2-3-fold de-
is conserved in SWL) and P9 in receptor binding. The alanine
6690 Biochemistry, Vol. 49, No. 31, 2010Mitra et al.
scanning approach did not provide information about the impor-
peptide (Figure 1A). However, its replacement in the YSA pep-
tide with a serine dramatically decreased potency (YSS-12mer in
remaining five residues of YSA to alanine did not substantially
affect the inhibitory potency of the peptide, suggesting that these
and/or stability). The identification of amino acids that are not
the peptide if a modified YSA peptide containing alanines at seve-
ral positions simultaneously retains high binding affinity. The 12
peptides used for alanine scanning were also examined for their
ability to inhibit binding of ephrin-A5 AP to the immobilized
detectable inhibitory activity for these other EphA receptors at a
concentration of 100 μM (data not shown), suggesting that the
single-alanine replacements do not generate less selective peptides
able to efficiently target other EphA receptors.
Molecular Docking of the YSA Peptide in the Ephrin-
Binding Channel of the EphA2 Receptor. Thestructureofthe
recently determined by X-ray crystallography, revealing the molec-
G-H loop and the EphA2 hydrophobic channel (35). We took
advantage of the information obtained from the alanine scan to
construct a model of the YSA peptide in complex with the EphA2
ligand-binding domain by using ZDOCK and RDOCK (37). We
defined residues Y1, Y4, P5, and D6 of the YSA peptide as impor-
tant for receptor binding, on the basis of the results of the alanine
scanning experiments. We also defined R103 of EphA2 as being
important for peptide binding because this residue was previously
shown to be critical for ephrin-A1 ligand binding (35).
the lowest RDOCK energy, the Y1, Y4, and D6 residues of the
Backboneatoms of YSA amino-terminalamino acidY1interact
withEphA2R132 andE157 through a hydrogenbond anda salt
bridge, respectively. The side chain hydroxyl group of Y4 in the
YSA peptide forms two hydrogen bonds with the backbone of
EphA2 V102 and K162. Consistent with the importance of the
hydroxyl group, changing Y4 to phenylalanine substantially
reduced the inhibitory potency of the peptide (YSAF in
Table 1). However, the peptide with alanine rather than pheny-
lalanine at position 4 did not have any detectable activity
(Figure 2A), suggesting additional interactions mediated by the
phenyl ring. In the crystal structure of the EphA2-ephrin-A1
complex, ephrin-A1 E119 forms a salt bridge with EphA2 R103,
which is essential for the binding of ephrin-A1 to EphA2 (35).
Interestingly, in the model, YSA D6 also interacts with EphA2
R103 through a hydrogen bond and two salt bridges. Thus,
overall the model is in good agreement with our experimental
evidence. However, it does not completely explain the substan-
Ala-1peptide,wherethebackboneinteractionsshould be mostly
preserved. This may be due to the fact that ZDOCK is a rigid
docking algorithm while EphA2 may assume a slightly different
conformation when bound to ephrin-A1 or the YSA peptide.
A structural alignment between the YSA peptide docked to
EphA2 and the ephrin-A1 G-H loop in the crystal structure of
the ephrin-A1-EphA2 complex (Figure 3C) suggests that YSA
P5 and ephrin-A1 P113 overlap and contribute to the formation
Thus, P5, which is also present in the SWL peptide (Figure 1A),
may help bend the peptide to fit in the EphA2 ephrin-binding
similar to that of the P113-F114 motif of the ephrin-A1 G-H
loop, which is conserved in all ephrin-A ligands except ephrin-
A3(38).Interestingly, thismotif alsoappearsto be presentinthe
KYL peptide, which targets the EphA4 receptor, and the KHL
and WASH peptides, which target the EphA7 receptor (38). The
other in spatial position, and they may interact with the same
EphA2 residues. YSA V8 overlaps with ephrin-A1 Phe111, and
FIGURE 2: Alanine scanfor the YSA peptide. (A) IC50values for the
indicated modified forms of the YSA peptide were calculated from
Fc. The table shows average IC50values, calculated from the indicated
number of experiments (n). Ala-1-Ala-12 are the peptides, where
accurately (>100 μM); for the Ala-4 peptide, no inhibition was
(.100 μM). (B) The histogram shows the IC50values ( the standard
error. The sequence of the YSA peptide is shown, with the residues
identified as critical for binding to EphA2 in bold.
Table 1: SAR for EphA2-Targeting Peptides
aResidues found only in the YSA peptide are colored black, residues found
not present in either of the original peptides green.
bNumber of experiments.
ArticleBiochemistry, Vol. 49, No. 31, 2010 6691
opposite amino- to carboxy-terminal orientations. Interestingly,
an opposite orientation was also previously observed for the
FSPN motif found in both the G-H loop of ephrin-B2 and the
TNYL-RAW peptide, both of which bind to the ligand-binding
channel of EphB4 and have been crystallized in complex with this
receptor (39, 40). Overall, our model of YSA in complex with
binding channel of EphA2. Furthermore, calculated docking
energies may be useful in prioritizing new YSA and SWL peptide
derivatives to be synthesized for further optimization.
YSA-9mer and YSA-5mer Peptides Retain Some of the
Binding Features of the YSA-12mer Peptide. The last three
amino acids of the YSA peptide did not seem to be critical for
binding to EphA2, based on their individual replacement with
alanine residues. This is consistent with the docking model of the
EphA2-YSA complex, where they are outside the ephrin-binding
pocket (Figure 3). We therefore synthesized a YSA-9mer peptide
lacking these amino acids. Besides being smaller, this peptide also
has an advantage in the fact that it does not contain methionine
residues, which can be easily oxidized (Table 1). The YSA-9mer
peptide exibited a 1.5-2-fold higher IC50value for inhibition of
ephrin-A5-EphA2 binding compared to the YSA-12mer peptide
in ELISAs (Figure 4A,B and Table 1). We also synthesized a
“minimal” YSA-5mer peptide comprising the five amino-terminal
residues of YSA and containing four of the five most critical
receptor binding determinants of the peptide. We found that the
5mer peptide retains the ability to bind to EphA2, albeit with an
affinity much lower than that of the YSA-12mer (Figure 4C).
Interestingly, despite its much reduced size, the YSA-5mer peptide
still retained high selectivity for EphA2 (Figure 4D). This suggests
that the (Y/W)XAYP motif, which is also conserved in the SWL
peptide but not in the ephrins, not only is critical for binding but
also may confer selectivity for EphA2.
Ephrin-A Fc fusion proteins have substantially increased
apparent affinity for EphA receptors because each molecule
has two EphA receptor binding sites (41). Dimeric peptides would
higher potency. We therefore synthesized a peptide corresponding
to the YSA-9mer with an additional carboxy-terminal cysteine
higher potency than the YSA-12mer, which was not substantially
affected by treatment with DTT to eliminate the possible inter-
molecular disulfide bond. Although unlikely under the conditions
of our experiment, we cannot exclude the possibility that the un-
paired cysteine in some of the peptide molecules may form a cova-
rized via a disulfide bond between the carboxy-terminal cysteines
and purified by HPLC, the dimeric peptide exhibited a further
3-fold increase in potency (Table 1). The small effect of dimeriza-
tion may in large part reflect the 2-fold higher concentration of
peptide moieties due to the dimeric nature of the molecules,
suggesting that longer linkers may be needed to avoid steric
hindrance. It will be interesting in future experiments to examine
of the YSA-9mer peptide through dimerization.
Potency and Stability of the YSA and SWL Peptides
and Their Derivatives. Because a number of residues are con-
served between the YSA-12mer and the SWL-12mer peptides
(Figure 1A),wegeneratedseveral hybrid peptides and character-
ized their ability to inhibit EphA2-ephrin-A5 interaction. For
with the “SWL” sequence, to generate the SWLA-13mer hybrid
FIGURE 3: Docking of the YSA peptide in the EphA2 ephrin-binding
by RDOCK. All top nine poses adopt a similar conformation in the
ephrin-binding channel of EphA2. The pose with the lowest RDO-
CK energy is presented as a stick structure (C colored green, O colored
line structures (C colored purple, O colored red, N colored blue, and S
colored yellow). The four amino acids found to be most critical for
EphA2 binding based on the alanine scan (Y1, Y4, P5, and D6) are
sentation, colored as in panel A) that is buried in the EphA2 channel
(presentedasa solidredribbonwithselectedresiduesthat arepredicted
bonds are represented by green dashed lines and salt bridges by black
lines (distances in angstroms and indicated by orange numbers).
(C) Comparison of the best docked conformation of the YSA peptide
6692 Biochemistry, Vol. 49, No. 31, 2010Mitra et al.
(Table 1). Similar to the YSA-12mer peptide, removal of the
three most carboxy-terminal amino acids of the SWLA-13mer
peptide (to generate the SWLA-10mer peptide) decreased po-
tency, while further addition of a carboxy-terminal cysteine
increased potency even when the peptide was treated with DTT
toeliminate the possible intermolecular disulfide bond (Table 1).
However, the SWL-12mer peptide was the most potent of the
peptides tested (Table 1). This is in contrast to our previous
results with YSA and SWL peptides that contained a carboxy-
terminal biotinylated GSGSK linker, which had KDvalues of
∼200 and ∼700 nM, respectively (29). It appears that the linker
increased the inhibitory potency of YSA but not SWL, based on
inhibition of ephrin-A5 AP binding to EphA2 Fc (compare
Figure 3A in ref 29 with Figure 4B and Table 1). Notably, in our
phage display screens, we isolated many more phage clones
displaying the SWL peptide than the YSA peptide (29). In
summary, our structure-activity relationship analysis reveals
that the SWL-12mer and dimerized YSA-C-10mer peptides are
the most potent EphA2-targeting peptides in the series examined
so far, followed by the SWLA-C-11mer peptide. However, the
FIGURE 4: YSA-9merpeptidethatretainsasubstantiallyhighpotencyandYSA-5merpeptidethatretainsahighselectivelyforEphA2.(AandB)
Binding of the indicated Eph receptor Fc fusion proteins, and ephrin-A5 Fc as a negative control, to the immobilized YSA-5mer peptide.
FIGURE 5: Stability of EphA2-targeting peptides. The indicated peptides were incubated in PC3 conditioned medium for 5 days at 37 ?C (with
ArticleBiochemistry, Vol. 49, No. 31, 20106693
unpaired cysteine in this peptide may result in some unwanted
reactivity (see above).
We also examined the stability of the YSA-12mer, SWL-12mer,
SWLA-13mer, and SWLA-C-11mer peptides in conditioned cell
culture medium from PC3 prostate cancer cells. We found that
these peptides are remarkably stable and are only slightly less
day incubation in the conditioned medium (Figure 5). In compar-
ison, a control peptide examined in parallel was completely inacti-
vated within a few hours (data not shown). This apparent resis-
tance to proteasesisconsistent with therecentlyreported abilityof
the YSA peptide to remain functional in culture medium and
ascites fluids (30-33).
The EphA2-Targeting Peptides Induce Receptor Phos-
phorylation and Inhibit Oncogenic Pathways in Cancer
Cells. We have previously shown that the YSA peptide with a
GSGSK-biotin tail can promote EphA2 tyrosine phosphory-
lation (which is indicative of activation) and induce biological
endothelial cells and COS cells (29, 42). We report here that the
SWL-12mer and SWLA-C-11mer peptides are more effective than
YSA-12mer in stimulating EphA2 tyrosine phosphorylation in
PC3 prostate cancer cells, consistent with their higher potency
(Figure 6A and Table 1). YSA-C-10mer also induced EphA2
phosphorylation (data not shown). Furthermore, the SWL-12mer
induced phosphorylation of Erk1/Erk2MAP kinases and Akt,
to that of ephrin-A1 Fc (13, 43) and suggests that the peptides can
and Ras-MAP kinase pathways, through activation of EphA2
(Figure 6B). Cell contraction, involving retraction of the cell
periphery and cell rounding, is another consequence of ligand-
12mer peptides induce PC3 cell contraction (Figure 6C). This
“repulsive” effect of EphA2 has been shown to involve weakened
β1-integrin-dependent cell substrate adhesion and RhoA-depen-
be monomeric, can function as agonists is consistent with the fact
that monomeric ephrin-A ligands havealso been shown to activate
EphA2 (46, 47). It will be interesting to determine whether ephrins
and peptides cause EphA2 activation by disrupting homophilic
interactions that involve the ephrin-binding channel. Such homo-
philic interactionshavebeen recentlyhypothesizedfrom the crystal
structure of the EphA2 ephrin-binding domain (35). Regardless of
the precise mechanism, peptides of the YSA/SWL series may have
some intrinsic tumor suppressor activities, in addition to being
possible tumor-targeting agents.
In conclusion, we have characterized a series of related
peptides that selectively bind to the EphA2 receptor to identify
features that are important for receptor binding. The peptides
include the YSA and SWL peptides identified by phage display,
derivatives of the YSA peptide with single-amino acid substitu-
tions, shortened forms of the peptides, a dimerized form, and
FIGURE 6: Biological effects of EphA2-targeting peptides. (A) PC3 cells were treated for 20 min with 0.1 μg/mL ephrin-A1 Fc and the indicated
antibodies and reprobedwithanti-EphA2antibodies. (B) Serum-starved PC3 cells were either leftuntreated (control),treated with10% FBS (in
duplicate samples), or treated with FBS together with 1 μg/mL ephrin-A1 Fc or the indicated peptides at 50 μM. Cell lysates were probed with
6694 Biochemistry, Vol. 49, No. 31, 2010Mitra et al.
hybrids of the two peptides. We found that the previously less
characterized SWL peptide is a viable alternative to the YSA
peptide and in fact appears to have a higher potency in its
unmodified form and lacks potentially problematic methionines.
The amino-terminal portion of the two peptides appears to be
the most important for receptor binding and selectivity, and
shorter peptides containing this region can be dimerized to
improve potency. In contrast, several amino acids are dispen-
sable for high potency and selectivity, particularly those in
the carboxy-terminal part of the peptides. Optimized peptides
of the YSA/SWL series could be used as starting points for
agents. An advantage of these peptides is that it will be straight-
forward to prepare homogeneous peptide conjugates by intro-
ducing a desired reactive amino acid that can be used for
conjugation, and our results show that the carboxy terminus is
the region more amenable to modification without an effect
on potency. In contrast, antibodies contain many reactive
groups, and conjugation yields many different molecular species
that may have different targeting and pharmacokinetic pro-
perties. In addition, peptides can be easily synthesized and
purified and tend to be nonimmunogenic. As an added benefit,
peptides of the YSA/SWL series also demonstrate an intrinsic
ability to activate the EphA2 receptor and inhibit several
oncogenic pathways, including the Ras-MAP kinase and the
PI3 kinase/Akt pathways, and integrin activity. These results are
in cancer therapeutic strategies to target EphA2, a receptor
widely expressed not only in cancer cells but also in the tumor
Ala-1, Ala-4, YSA-5mer, and YSA-12mer peptides.
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