Evaluation of dimerization-inhibitory activities of cyclic peptides containing a β-hairpin loop sequence of the EGF receptor

Article abstract of DOI:10.1016/j.bmc.2012.08.013

Structure-activity relationships of cyclic peptides mimicking the β-hairpin structure of the 'dimerization arm' at residues 242-259 of the EGF receptor are examined. Cyclic peptides containing the arm head of the β-hairpin loop showed inhibitory activity toward the EGF receptor's dimerization. Cyclic peptides containing a Retro-Inverso sequence of the dimerization arm showed clear inhibitory effects on the dimerization in vitro and efficiently suppressed the proliferation of A431 cells, which abundantly express the EGF receptor on their surface. The effects at a specific hydrophobic site of the loop structure were expected to enhance the interactions with the receptor.

Full text of DOI:10.1016/j.bmc.2012.08.013

Bioorganic & Medicinal Chemistry 20 (2012) 5730–5737
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of dimerization–inhibitory activities of cyclic peptides containing
a b-hairpin loop sequence of the EGF receptor
Takaaki Mizuguchi a, Naho Ohara a, Mika Iida a, Ryunosuke Ninomiya a, Shinji Wada a, Yoshiaki Kiso a,
,
Kazuki Saito a,b, Kenichi Akaji a,
⇑
a Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
b Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationships of cyclic peptides mimicking the b-hairpin structure of the ‘dimerization
arm’ at residues 242–259 of the EGF receptor are examined. Cyclic peptides containing the arm head of
the b-hairpin loop showed inhibitory activity toward the EGF receptor’s dimerization. Cyclic peptides
containing a Retro-Inverso sequence of the dimerization arm showed clear inhibitory effects on the
dimerization in vitro and efficiently suppressed the proliferation of A431 cells, which abundantly express
the EGF receptor on their surface. The effects at a specific hydrophobic site of the loop structure were
expected to enhance the interactions with the receptor.
Received 11 July 2012
Revised 9 August 2012
Accepted 9 August 2012
Available online 17 August 2012
Keywords:
EGF receptor
Dimerization
Inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
Cyclic peptide
Retro-Inverso modification
A431 cell
1. Introduction
receptor trans-phosphorylates the tyrosine residue at the C-termi-
nal tail of the partner.16–18 The phosphorylation induces cell growth
The epidermal growth factor (EGF) receptor is a membrane-
spanning protein belonging to the ErbB family (ErbB1–4).1,2 The
receptor is closely involved in cell proliferation and differentiation,
and its overexpression or unregulated activation can lead to the
aberrant growth of cancer cells such as non-small cell lung cancer
(NSCLC) cells.3–6 Thus, it is a target of anti-cancer drugs such as
gefitinib (IressaÒ) which directly inhibits its intracellular tyrosine
kinase domain.7,8 Antibodies against the EGF receptor also have
been developed as anti-cancer agents; cetuximab (ErbituxÒ), for
example, is a monoclonal antibody recognizing the extracellular
ligand-binding region of the receptor.9–11 Although these agents
are effective as cancer therapeutics, their use has several draw-
backs including side effects12 and exorbitant costs.13,14 A new type
of inhibitor against the EGF receptor is eagerly anticipated for
improving the quality of life of cancer patients.
through activation of an intracellular signaling pathway.19,20
The extracellular structure of the dimerized EGF receptor has
been elucidated by X-ray crystallographic studies (PDB ID:
1IVO).21 The receptor’s extracellular region consists of four do-
mains (I–IV), with most of the interface region of the dimer com-
posed of domain II. A b-hairpin arm at residues 242–259 in
domain II is thought to be essential to maintaining the dimeric
structure (Fig. 1).21 Tyr246, Thr249, Tyr251, and Gln252 of the b-
hairpin arm form hydrogen bonds with Cys283, Asn86, Arg285,
and Ala286 of the counterpart, respectively, to tightly hold to-
gether the active dimer. Thus, the b-hairpin structure is known
as the ‘dimerization arm’, and is expected to exist in all ErbB recep-
tors (ErbB-1–4) including homo- and hetero-dimer receptors.22–25
Because of the key function of the ‘dimerization arm’ to hold to-
gether the receptor, peptide sequences of the arm have been used
as a template to design novel compounds with inhibitory activity
toward the functional dimer. Several compounds such as
Inherbin326 and NSC5645227 derived from the arm structure have
been reported. We also have developed a cyclic decapeptide
which mimics the loop structure of the arm, and
Activation of the EGF receptor is triggered by the binding of EGF
to the extracellular region followed by a conformational change
causing the receptor’s dimerization.15 The dimerization induces
intracellular autophosphorylation, in which a kinase domain of each
showed that it inhibits the dimerization of the EGF receptor.28 In
the present study, we examined the structure–activity relation-
ships of 1 in detail focusing on (i) the ring’s size by adding or
removing the corresponding amino acids of the arm, (ii) the ring’s
⇑
Corresponding author. Tel.: +81 75 595 4636; fax: +81 75 591 9900.
E-mail address: akaji@mb.kyoto-phu.ac.jp (K. Akaji).
Present address: Department of BioScience, Nagahama Institute of Bio-Science
and Technology, Tamura-cho, Nagahama, Shiga 526-0829, Japan.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.013

T. Mizuguchi et al. / Bioorg. Med. Chem. 20 (2012) 5730–5737
5731
Figure 1. Structure of the EGF receptor dimer (left) and the intermolecular hydrogen-bonds at the dimerization arm (right inset) (PDB ID: 1IVO). The extracellular region of
the receptor consists of four domains (I–IV) with the interface mostly occupied by domain II. Four representative hydrogen-bonds between Tyr246A and Cys283B, Thr249A
and Asn86B, Tyr251A and Arg285B, and Gln252A and Ala286B are shown in the inset (A and B at each residue indicate monomer A in blue and monomer B in green).
position by shifting two cysteine residues, (iii) the effect of adding
an extra sequence to the ring by elongating amino acids at the
N- or C-terminus, and (iv) the topology of the side-chain structure
by inverting the sequence and/or stereo-structure. The inhibitory
activity for receptor dimerization was assessed using membrane
extracts from human epidermal carcinoma A431 cells, which abun-
dantly express the EGF receptor on their surface.29
the role of the ring structure. Peptides 2–4 having a fairly large ring
are expected to have a loop with a b-hairpin-like structure, since
each disulfide bond is replaced at a site where the bond distance
is similar to that of the corresponding b-hairpin site determined
by X-ray crystallographic studies.21 Peptides 5–7 have a slightly
larger ring than 1; the differences are in the range of 1–2 amino
acids residues. Peptides 8 and 9 have a slightly smaller ring than
1 due to the removal of one or two residues. Peptides 10–12 also
have a smaller ring than 1 caused by a shift in the position of the
cysteine residue toward the inside of the ring structure to make
the ring more compact. 13 contains two disulfide bonds, a combi-
nation of 1 and 12, which would make the ring structure more
rigid.
The test peptides were produced by conventional Fmoc-based
solid-phase peptide synthesis using 2-chlorotrityl resin. Single
disulfide bonds were constructed by air-oxidation without diffi-
culty, and double disulfide bonds by successive oxidation with
air and iodine. To avoid the conversion of Met to Met(O) by iodine,
the second oxidation reaction by iodine was stopped within a min-
ute. The homogeneity of each peptide was confirmed by analytical
HPLC and high-resolution FAB-MS. Dimerization-inhibitory activi-
ties of the synthetic peptides were assessed using membrane ex-
tracts of A431 cells, according to published procedures28,30 with
minor modifications. Briefly, the ligand-induced dimer receptor
was covalently linked by a hydrophilic, membrane-impermeable
cross-linker, bis (sulfosuccinimidyl) suberate (BS3).31 The mono-
mer (ꢀ170 kDa) and dimer (ꢀ350 kDa) of the receptor were sepa-
rated by SDS–PAGE and detected by immuno-blotting with an anti-
EGFR antibody. The amount of monomer and dimer was quantified
by densitometry of the bands (Fig. S3). The inhibitory activity of
2. Results and discussion
2.1. Effects of the ring structure
To evaluate the effect of ring-size on the dimerization’s inhibi-
tion, twelve peptides (2–13) were designed (Fig. 2 A). Since most
of these peptides have Tyr246, Thr249, Tyr251, and Gln252 sup-
posed to be key residues in the b-hairpin arm structure, examining
their effects on receptor dimerization would provide insight into
10
l
M of 1 was used as a reference standard in each assay.
Amounts of the dimeric receptor in the presence of test peptides
are summarized in Figure 2 B in which the amount without any
peptide is taken as 1.0. As previously reported,28 1 decreased the
amount of the dimer by inhibiting its formation; 10 lM of 1 re-
duced the amount to 69% of the control. The amounts of dimer in
the presence of 2–4 having a large ring were similar to the amount
obtained with 1, and in the presence of peptides 5–7 having a
slightly large ring, a little higher (ꢀ80%). The results suggest that
the peptides with a fairly large ring have the flexibility to adjust
their structure similar to that of the dimerization arm to show
Figure 2. (A) Structure of the synthesized cyclic peptides (1–13). (B) Amounts of
the receptor dimer in the presence of the cyclic peptides. The amount of the dimer
without the peptide is taken as 1.0. The results are shown as the mean standard
deviation for at least three independent experiments performed in duplicate.
inhibitory activity toward the dimer’s formation, whereas
a
slightly large ring would have rigidity to hold the arm in a some-
what different structure.

5732
T. Mizuguchi et al. / Bioorg. Med. Chem. 20 (2012) 5730–5737
In contrast, peptides 8–12 with a smaller ring had little effect on
amino acids to the C-terminus abolished the inhibitory activity
(peptides 17–19). Thus, the addition of residues to the b-hairpin
structure had little effect on the inhibitory activity of the arm
mimics. These results suggest that the b-hairpin structure of 1
interacts closely with the dimer counterpart and the addition of
amino acid residues to the ring structure hinders the interaction
probably due to steric effects. Based on these results, the effects
of the side-chain topology of 1, both overall and at specific residues,
were then examined.
the receptor’s dimerization, which suggests that the small ring has
a major effect on the side-chain orientation necessary to maintain
the arm’s structure. 12 decreased the amount of the dimer to 90%,
which indicates that 32% of the inhibitory activity of 1 was recov-
ered (a 10% decrease in 12 vs 31% decrease in 1). The double cy-
clized peptide 13 showed more effective inhibitory activity; the
amount of dimer was reduced to 83%, indicating 65% of the inhib-
itory activity of 1 was recovered (a 20% decrease in 13 vs 31% de-
crease in 1). The positions of two cysteine residues of 12
correspond to Asn247 and Thr250 of the dimerization arm. Accord-
ing to crystallographic studies (Fig. 1), these two residues occupy
an arm head region in the dimerization interactions and a hydro-
gen bond is estimated to exist between the side-chains. Thus, the
specific small ring which can hold the arm head structure might
compensate for the negative effects due to the downsizing of the
ring structure to recover the minimum interactions necessary for
the dimer to form. The rigid structure of the double disulfide of
13 might work more effectively than 12 to maintain the necessary
arm head structure resulting in an increase in inhibitory activity.
Taken together, the results summarized in Figure 2 suggest that
the loop structure of the b-hairpin arm, especially its arm head,
is critical for the receptor dimerization induced by EGF-binding,
and thus, peptide mimics of this arm would have inhibitory effects
on the dimerization.
2.3. Effects of the side-chain structure
To evaluate the overall side-chain topology, a Retro-Inverso (RI)
form of 1, the peptide 20, was synthesized. This modification in-
volves a reversion of the peptide sequence accompanied by the
replacement of each L-amino acid with the corresponding D-amino
acid. RI-modified biologically active peptides have been found to
retain the recognition properties or biological activities of the par-
ent peptides, since a similar topography of side chain orientations
can be maintained.32,33 Similar or improved interactions of RI-
modified peptides with proteins or receptors have also been re-
ported.34,35 In addition, the introduction of
D-amino acids enhances
resistance to enzymatic degradation.36
For comparison, 21 modified only by a reversion of the original
sequence with -amino acids and 22 having the original sequence
of -amino acids were also synthesized. As summarized in Figure 4,
L
D
2.2. Effects of additional sequences in the ring structure
nearly the same amount (68%) of dimer was detected in the pres-
ence of the RI-modified peptide 20, which indicates that 20 has the
same inhibitory activity as 1. In contrast, 21 and 22 had little effect
on the receptor’s dimerization. These results strongly suggest that
the overall topology of the side-chains of 1 is critical to the inter-
actions at the dimerization arm.
The effects of substitutions at a specific residue were then
examined focusing on neighboring positions of the ring structure
of 1, since a shift of the disulfide positions inside the ring
completely abolished the inhibitory activity (Fig. 2 10 and 11).
Thus, 23 having Ala instead of Tyr246, a C-terminal residue next
to Cys245 in 1, and 24–28 having substitutions at Met253, an
To evaluate the effects of adding amino acid residues to the core
arm structure, 14–19 were synthesized (Fig. 3). Peptides 14–16
have an additional N-terminal sequence of the b-hairpin loop and
17–19 have an additional C-terminal sequence. As shown in Figure 3
B, 14 having an extra Met residue reduced the amount of dimer
slightly more than 1 (64% vs 69%), that is, the inhibitory activity
of 14 was slightly increased. Further addition to the N-terminus,
however, decreased the inhibitory effect as in 15 and 16. Adding
Figure 4. Structure of the synthesized cyclic peptides (20–22). Small characters in
the sequence indicate D-amino acids. (B) Amounts of the receptor dimer in the
presence of the cyclic peptides. The amount without the peptide is taken as 1.0. The
results are shown as the mean standard deviation for at least three independent
experiments performed in duplicate.
Figure 3. (A) Structure of the synthesized cyclic peptides (14–19). (B) Amounts of
the receptor dimer in the presence of the cyclic peptides. The amount of the dimer
without the peptide is taken as 1.0. The results are shown as the mean standard
deviation for at least three independent experiments performed in duplicate.

T. Mizuguchi et al. / Bioorg. Med. Chem. 20 (2012) 5730–5737
5733
Figure 5. (A) Structure of the synthesized cyclic peptides (23–28). (B) Amounts of
the receptor dimer in the presence of the cyclic peptides. The amount without the
peptide is taken as 1.0. The results are shown as the mean standard deviation for
at least three independent experiments performed in duplicate.
N-terminal residue next to Cys254, were synthesized. Peptides 24–
26 had a non-polar aliphatic side-chain structure (Leu, Nle, and Ala,
respectively) and 27 and 28, a polar neutral side-chain structure
(Met(O) and Gln).
The substitution of Tyr246 in 23 completely abolished the
inhibitory activity (Fig. 5) probably due to the loss of the hydrogen
bond at this site (see Fig. 1). In contrast, peptides substituted at po-
sition 253 decreased the amount of dimer in the range of 73–88%,
which indicates that the substitutions at this site maintained the
Figure 6. (A) Typical immunoblotting bands of the autophosphorylated EGF
receptor. (B) Inhibitory effects of the peptide 20 on the autophosphorylation.
EGF-stimulated autophosphorylation in the absence of the test peptide is taken as
1.0. Data represent the mean for two independent experiments performed in
duplicate.
70% at 10
same inhibitory effect at 10
l
M (Fig. 6). The parent peptide 1 showed nearly the
M as previously reported.28 The re-
l
inhibitory activity. Notably, the
c-branched structure (Leu) and
sults suggest that 20 suppresses the EGF-stimulated intracellular
autophosphorylation in living cells dose dependently, and would
have an inhibitory effect on cell proliferation.
planer amide structure (Gln) having nearly the same size as Met
maintained the inhibitory activity, while the short (Ala) or long ali-
phatic side-chain (Nle) and polar sulfoxide (Met(O)) structures
lowered or almost abolished the activity. The results suggest that
some hydrophobic area remained unoccupied at this site, which
would agree with previous X-ray crystallographic studies on the
2.5. Inhibitory effects on cell proliferation
To estimate the effect in vivo, the inhibitory activity of the RI-
modified peptide 20 in A431 cells was evaluated. For comparison,
that of 1 and Inherbin3, an inhibitory peptide of the EGF receptor
reported by Xu et al.26 was also examined. As shown in Figure 7
20 suppressed cell proliferation to 81% of that with vehicle (dis-
tilled water), more than did the parent peptide 1 (84%) and Inher-
bin3 (95%). Thus, the peptide 20 suppresses the dimer’s formation
and autophosphorylation, which leads to a reduction in cell
growth. The results can be explained in terms of the metabolic sta-
bility of the RI-modified peptide as well as its arm mimic.
EGF receptor’s interaction with TGF
a indicating that Met253 of
the dimerization arm participates in the hydrophobic interaction
in the complex.37
2.4. Inhibitory effects on EGF receptor autophosphorylation
The inhibitory effect of the RI-modified peptide 20 on autophos-
phorylation of the receptor was then evaluated using the intact
A431 cells. For comparison, the effect of 1 was also examined in
parallel. The cell-based assay was performed by immunoblotting
as described previously28 with minor modifications. In this assay,
an anti-EGFR [pY1068] ABfinityTM antibody, which specifically
recognizes the receptor phosphorylated at Tyr1068, was used as
a primary antiboby. Tyr1068 at the C-terminal tail of the intracel-
lular domain is an autophosphorylation site that allows binding of
Grb2 and activation of the Ras-Raf-ERK1/ERK2 signaling pathway,
which is deeply related to the cell proliferaction and
differentiation.38
3. Conclusion
Peptides containing a b-hairpin loop structure at residues 242–
259 of the EGF receptor can mimic a key function of the ‘dimeriza-
tion arm’ to fold the dimeric structure induced by EGF-binding, and
peptide mimics of this arm have inhibitory effects on the dimeriza-
tion. An arm head structure of the b-hairpin loop was found to be
critical for the receptor’s dimerization, since peptide mimics of this
small arm head showed inhibitory effects on the dimerization. The
In the presence of 20, autophosphorylation of the receptor was
decreased by approximately 45% at the concentration of 1 nM and

5734
T. Mizuguchi et al. / Bioorg. Med. Chem. 20 (2012) 5730–5737
(150 mg, 0.050 mmol) were added 2% (v/v) 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) and 2% (v/v) piperidine in DMF
(1.0 mL) and the mixture was stirred for 5 min at 25 °C then
washed with DMF (5ꢁ) and CHCl3 (2ꢁ). Fmoc-Gln(Trt)-OH
(74 mg, 0.12 mmol), 1-hydroxybenzotriazole (HOBt) (19 mg,
0.12 mmol), TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyl-
uronium tetrafluoroborate) (39 mg, 0.12 mmol), and DIPEA
(42 lL, 0.24 mmol) in DMF (2.0 mL) were added, the mixture was
stirred for 1 h at 25 °C, and the resin was washed with DMF (5ꢁ).
Successive condensation of the corresponding Fmoc amino acid
derivatives was carried out by repeating the same deprotection/
coupling protocol. The N-terminal Fmoc-Cys(Trt)-OH (71 mg,
0.12 mmol) was condensed using DIC (diisopropylcarbodiimide)
(19 lL, 0.12 mmol) and HOBt (19 mg, 0.12 mmol) in DMF
(2.0 mL) at 25 °C for 1 h. The protected peptide-resin (84 mg)
was treated with trifluoroacetic acid (TFA)-ethandithiol-triisopro-
pylsilane-H2O (94.5:2.5:1:2.5, 2.0 mL) at 25 °C for 90 min. The
mixture was filtered and the filtrate was concentrated in vacuo.
Cold diethyl ether (30 mL) was added to the residue. The product
was suspended in distilled H2O and lyophilized to give 19 mg
(27%) of crude product. HPLC retention time (rt), 22.96 min
[YMC-Pack ODS-AM (4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–
100%)/100 min], MALDI-TOF-MS; found 1114.1617 for [M+Na]+,
calcd 1114.3950 for C46H65N11O16S2Na.
Figure 7. Inhibition of A431 cell proliferation. A431 cells were seeded in DMEM
(containing 10% FBS and antibiotics) and treated with 1, 20, and Inherbin3. Cell
growth with distilled water instead of the peptide was used as a 100% cell growth
control. Data represent the mean standard deviation for three independent
experiments performed in triplicate.
RI-modified peptide showed a similar level of inhibitory activity to
the parent peptide 1 in vitro and suppressed the proliferation of
A431 cells more effectively than 1, which strongly suggests that
the overall topology of the side-chains plays an important role in
the interactions of the arm mimics with the receptor. The side-
chain structure at Met253 might be a specific hydrophobic site that
enhances interaction with the receptor. Further modifications
focusing on the side-chain interactions are currently underway in
our laboratory.
The crude peptide (12 mg) in an ammonium bicarbonate buffer
(120 mL, pH 8.1, peptide concentration of 0.1 mg/mL) was stirred
in air at 25 °C. An aliquot (50 lL) was subjected to analytical re-
versed-phase HPLC [YMC-Pack ODS-AM (4.6 ꢁ 150 mm), 40 °C,
0.1% TFA/CH3CN (0–100%/100 min) in H2O, 0.9 ml/min]. After
10 h, the solution was lyophilized to yield a white powder contain-
ing ammonium bicarbonate (70 mg): MALDI-TOF-MS; found
1112.3088 for [M+Na]+, calcd 1112.3793 for C46H63N11O16S2Na.
The product was purified by preparative RP-HPLC [YMC-Pack
ODS-AM (20 ꢁ 250 mm), 40 °C, 0.1% TFA/CH3CN (15–55%/80 min)
in H2O, 5 ml/min]. Yield 2.3 mg (19%). HPLC rt, 18.91 min (single
peak) [YMC-Pack ODS-AM (4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN
(0–50%)/50 min], FAB-MS; found 1112.3800 for [M+Na]+, calcd.
1112.3793 for C46H63N11O16S2Na.
4. Experimental
4.1. General
The Fmoc amino acid derivatives, Asp(t-Bu), Glu(t-Bu), Thr(t-
Bu), Tyr(t-Bu), Lys(Boc), Trp(Boc), Cys(Trt), Cys(Acm), Asn(Trt),
and Gln(Trt), were purchased from Calbiochem-Novabiochem Ja-
pan Ltd. (Tokyo, Japan) or Watanabe Chemical Ind., Ltd (Hiroshima,
Japan). The human epidermal carcinoma A431 cells were from
American Type Culture Collection (ATCC; Manassas, VA), and fetal
bovine serum (FBS) from Thermo Fisher Scientific Inc. (Rockford,
IL). The reagents were purchased from Nacali Tesque (Kyoto, Japan)
and used without further purification.
Analytical HPLC was performed using a C18 reversed phase col-
umn (4.6 ꢁ 150 mm; YMC Pack ODS AM) at 40 °C with a binary sol-
vent system; a linear gradient of CH3CN in 0.1% aqueous TFA at a
flow rate of 0.9 mL/min with detection at 230 nm. Preparative
Other cyclic peptides containing a single disulfide bond were
similarly synthesized and purified. All test compounds showed
P95% purity (Fig. S2).
peptide 2
HPLC rt, 35.00 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min, (50–100%)/
5 min], FAB-MS; found 2405.9902 for [M+Na]+, calcd. 2405.9891
for C103H154N24O33S4Na.
peptide 3
HPLC was carried out using
a C18 reversed phase column
HPLC rt, 33.28 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 2154.8613 for [M+Na]+, calcd. 2154.8621 for
(20 ꢁ 250 mm; YMC Pack ODS AM) at 40 °C with a binary solvent
system; a linear gradient of CH3CN in 0.1% aqueous TFA at a flow
rate of 5 mL/min with detection at 230 nm. The solvents were of
HPLC grade. MALDI-TOF-MS spectra were recorded on a Voyager
C91H137N21O30S4Na.
DE-STR using a-cyano-4-hydroxy cinnamic acid as a matrix. High
resolution FAB-MS spectra were obtained on a JEOL JMS-SX102A
spectrometer.
peptide 4
HPLC rt, 31.00 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1701.6405 for [M+Na]+, calcd. 1701.6397 for
4.2.
8
Cys-Tyr-Asn-Pro-Thr-Thr-Tyr-Gln-Cys
C71H106N16O23S4Na.
First, 400 mg (0.52 mmol) of 2-chlorotrityl chloride resin
(1.3 mmol/g) was agitated in CH2Cl2 (3.0 mL) at 25 °C for 10 min.
Next, Fmoc-Cys(Trt)-OH (150 mg, 0.26 mmol) and diisopropyleth-
ylamine (DIPEA) (91 lL, 0.52 mmol) were added and the mixture
was stirred for 150 min at 25 °C. The resin was washed with
peptide 5
HPLC rt, 26.53 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1471.5305 for [M+Na]+, calcd. 1471.5308 for
C61H88N14O21S3Na.
DMF, CHCl3, and MeOH, and dried in vacuo. To the resulting resin

T. Mizuguchi et al. / Bioorg. Med. Chem. 20 (2012) 5730–5737
5735
peptide 6
HPLC rt, 22.41 min (single peak) [YMC-Pack ODS-AM
peptide 19
HPLC rt, 25.33 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1682.6271 for [M+Na]+, calcd. 1682.6265 for
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1358.4464 for [M+Na]+, calcd. 1358.4468 for
C55H77N13O20S3Na.
C70H101N17O24S3Na.
peptide 7
HPLC rt, 27.18 min (single peak) [YMC-Pack ODS-AM
peptide 20
HPLC rt, 21.80 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1243.4202 for calcd 1243.4198 for
[M+Na]+,
51H72N12O17S3Na.
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1356.5045 for [M+Na]+, calcd. 1356.5039 for
C57H83N13O18S3Na.
C
peptide 9
HPLC rt, 20.31 min (single peak) [YMC-Pack ODS-AM
peptide 21
HPLC rt, 21.59 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1243.4203 for [M+Na]+, calcd. 1243.4198 for
C51H72N12O17S3Na.
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 984.3202 for [M+Na]+, calcd. 984.3208 for C41H55N9O14S2Na.
peptide 10
HPLC rt, 19.21 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1227.4059 for [M+Na]+, calcd. 1227.4063 for
peptide 22
HPLC rt, 22.31 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1243.4196 for [M+Na]+, calcd. 1243.4198 for
C51H72N12O17S3Na.
C50H68N12O19S2Na.
peptide 11
HPLC rt, 21.25 min (single peak) [YMC-Pack ODS-AM
peptide 23
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
HPLC rt, 20.22 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1151.3933 for [M+Na]+, calcd. 1151.3936 for
found 1177.4274 for [M+Na]+, calcd. 1177.4270 for
C47H70N12O18S2Na.
C45H68N12O16S3Na.
peptide 12
HPLC rt, 25.27 min (single peak) [YMC-Pack ODS-AM
peptide 24
HPLC rt, 24.85 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1256.4397 for [M+Na]+, calcd. 1256.4402 for
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
C53H75N11O17S3Na.
found 1225.4641 for [M+Na]+, calcd. 1225.4634 for
C52H74N12O17S2Na.
peptide 14
HPLC rt, 25.75 min (single peak) [YMC-Pack ODS-AM
peptide 25
HPLC rt, 24.97 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1225.4637 for [M+Na]+, calcd. 1225.4634 for
C52H74N12O17S2Na.
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1374.4609 for [M+Na]+, calcd. 1374.4603 for
C56H81N13O18S4Na.
peptide 15
HPLC rt, 28.22 min (single peak) [YMC-Pack ODS-AM
peptide 26
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
HPLC rt, 19.14 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1183.4166 for [M+Na]+, calcd. 1183.4165 for
found 1487.5441 for [M+Na]+, calcd. 1487.5444 for
C62H92N14O19S4Na.
C49H68N12O17S2Na.
peptide 16
HPLC rt, 30.65 min (single peak) [YMC-Pack ODS-AM
peptide 27
HPLC rt, 19.40 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1584.5978 for [M+Na]+, calcd. 1584.5971 for
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
C67H99N15O20S4Na.
found 1259.4143 for [M+Na]+, calcd. 1259.4147 for
C51H72N12O18S3Na.
peptide 17
HPLC rt, 27.15 min (single peak) [YMC-Pack ODS-AM
peptide 28
HPLC rt, 19.28 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1342.4875 for [M+Na]+, calcd. 1342.4882 for
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
C56H81N13O18S3Na.
found 1240.4377 for [M+Na]+, calcd. 1240.4379 for
C51H71N13O18S2Na.
peptide 18
HPLC rt, 24.75 min (single peak) [YMC-Pack ODS-AM
4.2.26. H-Leu-Val-Tyr-Asn-Lys-Leu-Thr-Phe-Gln-Leu-Glu-Pro-
Asn-Pro-His-Thr-Lys-OH (Inherbin3)
HPLC rt, 28.37 min (single peak) [YMC-Pack ODS-AM
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
(4.6 ꢁ 150 mm), 0.9 ml/min, CH3CN (0–50%)/50 min], FAB-MS;
found 1456.5309 for [M+Na]+, calcd. 1456.5312 for
C60H87N15O20S3Na.

5736
T. Mizuguchi et al. / Bioorg. Med. Chem. 20 (2012) 5730–5737
found 2064.0891 for [M+Na]+, calcd. 2064.0894 for
95H148N24O26Na.
4.5. EGF receptor autophosphorylation assay
C
A431 cells in the DMEM supplemented with 10% FBS and 1%
4.3.
13
penicillin–streptomycin (v/v; 10,000 units/ mL and 10,000 lg/
Cys-Tyr-Cys-Pro-Thr-Cys-Tyr-Gln-Met-Cys
mL, respectively) were seeded at 1 ꢁ 104 cells per well in 24-well
plates and allowed to grow at 37 °C for 4 days in the incubator
under 5% CO2. After starvation in serum-free medium containing
1 mg/mL of BSA (bovine serum albumin) at 37 °C for 24 h, the cells
were treated with the peptide 20 or PBS at 37 °C for 1 h. They were
then treated with 100 ng/mL of human EGF (recombinant, R&D
Systems, Inc., Minneapolis, MN) at 37 °C for 5 min, put on ice and
quickly washed three times with cold PBS. The cells were solubi-
lized on ice for 10 min with a lysis buffer containing 50 mM HEPES
(pH 7.4), 1% (v/v) NP-40, 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v)
Protease Inhibitor Cocktail (100ꢁ) (Nacalai Tesque, Inc.) and 1% (v/
v) Phosphatase Inhibitor Cocktail (100ꢁ) (Nacalai Tesque, Inc.). To
remove the cell debris, the extracts were centrifuged at 14,000 ꢁ g
for 10 min at 4 °C, and the protein concentrations of the whole-cell
lysate were measured with a BCA™ Protein Assay kit (Thermo Fish-
er Scientific Inc.). The supernatant was diluted with the lysis buffer
H-Cys-Tyr-Cys(Acm)-Pro-Thr-Cys(Acm)-Tyr-Gln-Met-Cys-OH
130 was synthesized as described above. A solution of the crude
peptide 130 (11 mg) in an ammonium bicarbonate buffer (50 mL,
pH 8.0) was stirred in air at 25 °C, and the reaction was monitored
as above. After 47 h, the solution was lyophilized to yield 10 mg
(91%) of a cyclized peptide 1300: MALDI-TOF MS; found 1376.24
for [M+Na]+, calcd 1376.45 for C55H79N13O17S5Na. The obtained
peptide 1300 (3.1 mg) was mixed with 0.1 M I2 in 50% AcOH
(2.5 mL, 100 equiv) and the solution was stirred for 1 min at
25 °C. Two drops of 1 M ascorbic acid solution in H2O were added
and the solution was concentrated in vacuo. The product was puri-
fied by preparative RP-HPLC as above to yield 0.36 mg (12%) of 13
as a white amorphous powder: HPLC rt, 25.13 min (single peak)
[YMC-Pack ODS-AM (4.6 ꢁ 150 mm), 0.9 ml/ min, CH3CN (0–
50%)/50 min], FAB-MS; found 1232.3312 for [M+Na]+, calcd.
1232.3319 for C49H67N11O15S5Na.
to 500 lg/ mL of protein. An aliquot was mixed with DTT and Nu-
PAGEÒ LDS Sample Buffer (4ꢁ) (Life Technologies Corporation),
and the mixture was boiled at 85 °C for 5 min. The product was
separated by SDS-PAGE (NuPAGEÒ 4–12% Bis-Tris Gel, Life Technol-
ogies Corporation) and proteins were transferred onto a PVDF
membrane. The membrane was treated with Blocking One-PÒ
(Nacalai Tesque, Inc.) at 25 °C for 30 min, washed twice with
TBS-T containing tris(hydroxymethyl)aminomethane (2.4 g/L),
NaCl (8.0 g/L), and 0.1% (v/v) Tween 20 for 5 min, and incubated
overnight with anti-EGFR [pY1068] ABfinityTM Recombinant rabbit
monoclonal Antibody-Purified (4 lL) (Life Technologies Corpora-
tion) in TBS-T (25 mL) at 25 °C. The membrane was washed three
times in TBS-T for 10 min, incubated with HRP-conjugated anti-
rabbit IgG antibody (2.5 lL) (NA934, GE Healthcare Ltd.) in TBS-T
(25 mL) at 25 °C for 1 h, and washed three times in TBS-T for
10 min. Phosphorylated bands of the EGF receptor were visualized
by the AmershamTM ECL Prime system (GE Healthcare Ltd), and
intensities were quantified with a Luminescent Image Analyzer,
LAS-4000mini. Data represent the mean for two independent
experiments performed in duplicate. To determine the receptor
amounts loaded in each lane of the SDS-PAGE gel, the blotted
membrane was treated with Stripping Solution (Wako Pure Chem-
ical Ind., Ltd, Osaka, Japan) at 25 °C for 10 min, and re-probed with
a rabbit polyclonal anti-EGFR antibody (1005) in combination with
a peroxidase-linked anti-rabbit IgG secondary antibody (NA934).
4.4. Inhibition assay for EGF receptor dimerization
A431 cells were cultured in Dulbecco’s modified Eagle’s med-
ium containing 4.5 g/L glucose,
(DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin–streptomycin (v/v; 10,000 units/ mL and 10,000 g/mL,
L-glutamine, and sodium pyruvate
l
respectively) at 37 °C under a humidified atmosphere of 5% CO2.
The cells (confluent in a 10-cm dish) were washed five times with
PBS, collected with a cell scraper, and centrifuged at 2300 ꢁ g at
4 °C for 5 min. The pellets were solubilized for 10 min with 1 ml
of a buffer containing 20 mM HEPES (pH 7.4), 1% (v/v) Triton X-
100, 10% (v/v) glycerol, 1 mM EGTA, and 1 mM phenylmethylsul-
fonyl fluoride (PMSF). The extract was centrifuged at 250,000 ꢁ g
for 10 min at 4 °C, and the protein concentration of the superna-
tant was determined with a BCA™ Protein Assay kit (Thermo Fish-
er Scientific Inc.). An aliquot of the supernatant, diluted with the
solubilization buffer to 2
10 M of each test peptide for 60 min. After incubation with or
without 1.4 g/mL (0.21 M) of human EGF (recombinant, R&D
lg/lL protein, was pre-treated with
l
l
l
Systems, Inc., Minneapolis, MN, USA) for 10 min, the mixture
was treated with 1 mM BS3 (Thermo Fisher Scientific Inc.) for
10 min. Next, 50 mM of glycine was added to terminate the
cross-linking reaction. Dithiothreitol (DTT) and NovexÒ Tris-Gly-
cine SDS sample buffer (2ꢁ) (Life Technologies Corporation, Carls-
bad, CA, USA) were then added and the mixture was boiled at
85 °C for 5 min. The product was separated by SDS-PAGE and pro-
teins were transferred onto a PVDF membrane. The membrane
was treated with Blocking OneÒ (Nacalai Tesque, Inc.) at 25 °C
for 1 h, washed two times with PBS-T containing NaCl (8.0 g/L),
KCl (0.2 g/L), Na2HPO4ꢂH2O (2.9 g/L), KH2PO4 (0.2 g/L), and 0.1%
(v/v) Tween 20, and incubated overnight with anti-EGFR antibody
4.6. Cell growth assay
A431 cells in the DMEM supplemented with 10% FBS and 1%
penicillin–streptomycin (v/v; 10,000 units/ mL and 10,000 lg/mL,
respectively) were seeded at 5 ꢁ 103 cells per well in 96-well
plates and allowed to grow at 37 °C for 24 h in the incubator under
5% CO2. The cells were treated with distilled water or 10
peptide and re-incubated at 37 °C for 24 h for three consecutive
days. Cell count reagent SF containing WST-8 (10 L/well) was
lM of test
(4 lL) (sc1005; Santa Cruz Biotechnology, Inc., Santa Cruz, CA,
l
USA) in PBS-T (25 mL) at 25 °C. The membrane was washed three
times in PBS-T for 10 min, incubated with HRP-conjugated anti-
rabbit IgG antibody (0.5 lL) (NA934, GE Healthcare Ltd.) in PBS-
added to each well and the plate was incubated at 37 °C under
5% CO2 for 90 min. Absorption values at 450 nm (reference:
655 nm) were measured on a Microplate Reader SH-1000 Lab. Data
represent the mean standard deviation. Each experiment was
performed in six replicate wells and independently repeated three
times.
T (25 mL) at 25 °C for 1 h, and washed three times in PBS-T for
10 min. Dimeric and monomeric bands of the EGF receptor were
visualized by the Amersham ECL Plus system (GE Healthcare
Ltd., Buckinghamshire, UK), and intensities were quantified
with a Luminescent Image Analyzer, LAS-4000mini (FUJIFILM
Corporation, Tokyo, Japan). Data represent the mean standard
deviation for at least three independent experiments performed
in duplicate.
Acknowledgments
This study was supported in part by the 21st Century COE
Program and the ‘Academic Frontier’ Project for Private Universi-

T. Mizuguchi et al. / Bioorg. Med. Chem. 20 (2012) 5730–5737
5737
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Supplementary data
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