Discovery of thioether-bridged cyclic pentapeptides binding to Grb2-SH2 domain with high affinity

Article abstract of DOI:10.1016/j.bmcl.2009.03.134

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor-bound protein 2 (Grb 2) is considered to be an effective and non-cytotoxic strategy to develop new ant

Full text of DOI:10.1016/j.bmcl.2009.03.134

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2693–2698
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of thioether-bridged cyclic pentapeptides binding to Grb2-SH2
domain with high affinity
b
c
a
c
c
Sheng Jiang a, , Chenzhong Liao , Lakshman Bindu , Biaolin Yin , Karen W. Worthy , Robert J. Fisher ,
*
Terrence R. Burke Jr. b, Marc C. Nicklaus b, Peter P. Roller b
a Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, PR China
b Laboratory of Medicinal Chemistry, National Cancer Instistute, NIH, Frederick, MD 21702, USA
c Laboratory of Protein Chemistry, SAIC-Frederick, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src
homology 2 (SH2) domain of the growth factor receptor-bound protein 2 (Grb 2) is considered to be
an effective and non-cytotoxic strategy to develop new anti-proliferate agents due to its potential to shut
down the Ras activation pathway. In this study, a series of phosphotyrosine containing cyclic pentapep-
tides were designed and synthesized based upon the phage library derived cyclopeptide, G1TE. A compre-
hensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this
novel template. With both the peptidomimetic optimization of the amino acid side-chains and the con-
straint of the backbone conformation guided by molecular modeling, we developed several potent antag-
Received 15 December 2008
Revised 6 March 2009
Accepted 26 March 2009
Available online 29 March 2009
Keywords:
Grb2-SH2 domain
Cyclic peptides
G1TE analogs
Phosphotyrosine
Grb2-SH2 antagonist
onists with low micromolar range binding affinity, such as cyclic peptide 15 with an Kd = 0.359 lM,
which is providing a novel template for the development of Grb2-SH2 domain antagonists as potential
therapeutics for certain cancers.
Ó 2009 Elsevier Ltd. All rights reserved.
Growth factor receptor-bound protein 2 (Grb2) is an essential
adapter protein, possessing a single Src homology 2 (SH2) domain
flanked by two SH3 domains.1 Grb2-SH2 domain directly mediates
erbB-2 (HER-2/neu) signaling by binding to pTyr motifs and the
downstream activation of mitogenic Ras pathways, which have
been implicated in the etiology of certain breast cancer.2,3 Grb2-
SH2 domain is an ideal target for drug development,4,5 in that
the phosphopeptide ligands that bind it are required to adopt a
b-turn conformation, which has been demonstrated by both NMR
and X-ray analysis.6–8 Molecules that can selectively antagonize
the Grb2-SH2 should interrupt these signaling pathways. These
agents may act as therapeutic leads for diseases, such as cancer,
in which the Ras signaling pathway plays a major role.9–12
ther-cyclized analogs were developed, some of which exhibited potent
binding affinity.14–19 G1TE and its analogues may form a largest circle-
like binding surface when they bind to Grb2-SH2 domain, as suggested
by molecular modeling and NMR studies.20,21 Molecular modeling of
the complex structure of Grb2-SH2 protein with G1TE indicated that
the side chains of the amino acid residues in the 1, 3 and 5 positions
of G1TE form strong hydrogen-bond interactions with the Arg 67
(aA-helix) and Arg 86 (bC-strand) residues and the three Ser residues
88, 90, and 96, and Lys 109 and Leu 120 within the binding pocket.
However, the amino acid residues in the 2, 7, 8, and 9 positions of
G1TE do not interact with the Grb2-SH2 protein. To further improve
the affinity and reduce the peptide character of the novel Grb2-SH2 do-
main antagonists, wewereinterestedindeveloping anewscaffoldwith
smaller ring size but higher affinity. We assumed that keeping essential
amino acids and deleting unnecessary amino acids of G1TE would in-
crease the binding affinity and selectivity. Replacement of Glu1,
Leu2 and Tyr3 of G1TE with p-Tyr would increase hydrogen-bond
interaction with the Arg 67 and Arg 86 of the SH2 domain binding
cavity; on the other hand, the phosphoryl group itself would un-
dergo additional intramolecular hydrogen-bond interactions, which
stabilizes the binding conformation of cyclic peptide. This is a
brand new strategy distinct from any approaches reported previ-
ously to improve the selectivity and affinity. This article reports
our recent discovery of potent tyrosine-phosphorylated G1TE ana-
logues by overall side-chain and backbone modifications and con-
formational constraint (Fig. 1).
In our previous studies, we discovered a novel disulfide-bridged
cyclic peptide G1, generated by phage library.13 This peptide was
comprised of the sequence -Glu-Leu-Tyr-Glu-Asn-Val-Gly-Met-
Tyr-, bracketed by two terminal cysteines that formed the cyclic
peptide through
a disulfide linkage. Subsequently, we further
designed and synthesized its redox stable thioether-bridged analogue,
cyclo (CH2CO-Glu1-Leu2-Tyr3-Glu4-Asn5-Val6-Gly7-Met8-Tyr9-Cys10)-
amide, termed G1TE (Fig. 1), which has shown comparable binding
affinities(IC50 = 15–20 lM)totheGrb2-SH2domainprotein, inBiacore
binding assays.13 Based on the G1TE, a number of redox-stable thioe-
* Corresponding author. Tel.: +86 20 32290439; fax: +86 20 32290606.
E-mail addresses: jiang_sheng@gibh.ac.cn, jiangsh9@gmail.com (S. Jiang).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.134

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S. Jiang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2693–2698
O
O
S
NH
O
Replacement with pTyr
OH
O
HN
HN
HN
NH2
OH
P
O
O
Ach
O
O
OH
Acp
OH
Atp
O
O
HO
O
H
H
N
O
N
2
NH
OH
NH
S
1
N
H
3
4
O
O
O
O
O
HN
O
N
H
NH
5
S=O
S
G1TE
O
O
H2N
O
S
1
O
NH
NH2
O
NH2
O
HN
6
HN
O
H
N
10
N
9
HN
O
O
H
HN
O
O
Cys(O)-(R)
S=O
H2N
8
HN
HN
O
7
O
OH
HN
OH
deletion
O
Cys(O)-(S)
NH
HN
Tyr
HN
Trp
Figure 1. Molecular design of Grb2-SH2 domain antagonists based upon thioether-bridged cyclic peptide G1TE.
HN
O
O
O
2-NaI
The cyclic peptides 1–15 in Table 1 were synthesized by using
by using NMM as a base. The resulting cyclopeptide bound resins
were washed six times with NMP, DCM, and MeOH, and then dried
under reduced pressure overnight. The cyclopeptides were cleaved
from the resin and fully deprotected by treating with 2.5% TIS and
2.5% H2O in TFA (10 mL/g resin) for 2 h. The oxidation of the thio-
ether functionality into sulfoxide was accomplished using a 5%
H2O2 aqueous solution. The resulting two isomers were easily sep-
arated by RP-HPLC. All peptides were purified to homogeneity by
preparative RP-HPLC (Vydac C18 column), and their structures
were confirmed by MALDITOF-MS mass spectrometry. The abso-
lute configuration of the sulfoxide diastereoisomers was deter-
mined by CD spectra using documented method.22–26
the procedure described in Fig. 2. The linear peptide precursors
were synthesized on an ABI 433A peptide synthesizer, starting
with Rink Amide resin for establishing the C-terminal carboxam-
ide, and using the chemical protocols based on the Fmoc chemistry.
The incorporation of N-Fmoc-Tyr[PO(OH, OBzl)-OH was carried out
manually and coupled twice using HATU as the coupling reagent.
After completion of peptide elongation, the Mmt protecting group
on Cys was removed by treating with 1% TFA in DCM to afford the
resin bound peptide 19. Subsequently, cyclic peptides were formed
The synthesis of cyclic peptide 16 was described in Fig 3. The
linear peptide precursors H-(Boc)Lys-pTyr(OBzl)-Ach-Asn-Val-2-
Cl-Trt resin were synthesized by Fmoc SPPS. After the peptides
were cleaved from resin, the head-to-tail macrocyclization of the
linear peptide was achieved successfully using HATU as the cou-
pling reagent and HOAt as the accelerator27 and then deprotected
with the TFA–TIS–H2O cocktail. After side-chain deprotection with
the TFA–TIS–H2O cocktail, the cyclic peptide 16 was obtained in
30% overall yield.
Docking studies of these 16 compounds were performed on the
crystal structure of Grb2-SH2 domain (PDBID: 1TZE),6 to which the
KPFpYVNV peptide bound, using the program Glide 4.5,28–31 part
of the First Discovery suite (Maestro). The starting structure for the
protein was prepared and fixed by the Protein Preparation Wizard,
a comprehensive protein preparation facility. All water molecules
were deleted; the bound ligand (KPFpYVNV) was adjusted manu-
ally; bond orders were assigned, and then hydrogen was added to
all atoms in the structure. The orientation of hydroxyl groups, amide
groups of Asn and Gln, and charge state of His residues were opti-
mized using the utility protassign. The final step was a restrained
minimization by the impref utility until the average root-mean-
square-deviation (rmsd) of the hydrogen atoms reached 0.3 Å,
leaving heavy atoms in place. In order to study the binding modes
of the antagonists, grids files representing shape and properties of
Table 1
Grb2-SH2 domain inhibitory activity of the G1TE analogs 1–16 with the general
structure cyclo-(pTyr1–AA2–Asn3–AA4–AA5)-amidea
b
Compds
Modifications of amino acid residues
Activity Kd
(
lM)
AA2
AA4
AA5
G1TE
1
2
3
4
5
6
7
8
—
—
—
25
Glu
Ach
Ach
Ach
Ach
Ach
Ach
Ach
Ach
Ach
Ach
Ach
Ach
Atp
Acp
Val
Val
Tyr
Trp
2-NaI
Val
Cys-CH2CO-
Cys-CH2CO-
Cys-CH2CO-
Cys-CH2CO-
3.6 ( 0.0.01)
1.42 ( 0.04)
1.06 ( 0.03)
1.05 ( 0.03)
2.71 ( 0.09)
1.56 ( 0.05)
1.76 ( 0.04)
1.36 ( 0.04)
1.72 ( 0.05)
2.14 ( 0.06)
1.29 ( 0.03)
4.8 ( 0.0.03)
ndc
Cys-CH2CO-
Cys(O)-(R)-CH3CO-
Cys(O)-(S)-CH2CO-
Cys(O)-(R)-CH3CO-
Cys(O)-(S)-CH2CO-
Cys(O)-(R)-CH3CO-
Cys(O)-(S)-CH2CO-
Cys(O)-(R)-CH3CO-
Cys(O)-(S)-CH2CO-
Cys-CH2CO-
Val
Tyr
Tyr
Trp
Trp
2-Nal
2-Nal
Val
9
10
11
12
13
14
15
0.88 ( 0.20)
0.359 ( 0.06)
Val
Cys-CH2CO-
16
Ach
Val
Lys
7.55
a
The binding affinity of these compounds was evaluated by Biacore assays.
b
Values are means of two experiments, standard deviation is given in
parentheses.
c
nd = not determined.

S. Jiang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2693–2698
2695
O
O
H
(BrCH2CO)2O
H2N
N
Peptide HN
Peptide HN
Br
N
H
N
H
DMF, r.t. overnight
O
Mmt
S
Mmt
18
S
17
O
O
H
N
Peptide HN
1% TFA in DCM
5%NMM in NMP
overnight
Peptide HN
N
H
Br
N
H
HN
S
2 mins, 10 times
O
HS
19
O
20
O
O
95% TFA, 2.5% TIS
and 2.5% H2O, 2hrs
Peptide HN
Peptide HN
NH2
NH2
5% H2O2
2 days
HN
S
HN
S
O
O
O
1-5, 14, 15
6-13
Figure 2. General procedure for the synthesis of thioether and sulfoxide bridged cyclic pentapeptides.
O
1% TFA in DCM
2 mins, 10 times
SPPS
NH2-Val-O
2-Cl-Trt Resin
21
(Boc)Lys-pTyr(OBzl)-Ach-Asn-
H2N
OH
HN
O
22
OH
P
O
P
O
OBzl
OH
O
O
O
O
NHBoc
HN
NH
NH2
(1) HATU/HOAt/DIPEA
in DCM
HN
N
H
N
H
O
O
O
O
O
(2) 95% TFA, 2.5% TIS
and 2.5% H2O, 2h
O
NH
O
O
O
O
NH2
OH
HN
HN
HN
H2N
H2N
23
16
Figure 3. Synthetic route of cyclic pentapeptide 16.
the receptor were generated. The three-dimensional structures of
the compounds 1–16 were constructed using the builder tool avail-
able through Maestro interface by modifying the known peptide
bound in 1TZE. The initial geometries of the structures were opti-
mized using the OPLS_2005 force field, performing 5000 steps of
conjugate gradient minimization. The compounds were subjected
to flexible docking using the pre-computed grid files. Glide 4.5 has
three docking precision mode: high-throughput virtual screening
(HTVS), standard precision (SP) and extra precision (XP). Here, to
try to get the closet binding conformations, we used XP mode.
To try to explain the biological activity differences between cyc-
lic peptides, conformational searches were adopted. The software
used was MACROMODEL 9.5,32 and the method utilized was Torsional
sampling (Monte Carlo Multiple Minimum, MCMM), with the
OPLS_2005 force field. Most experimental studies are carried out
in solvent, and the solvent medium can have a major effect on
molecular structures and energies, especially for molecules having
several polar functional groups, like those we study here. So when
doing conformation searching, we used a called GB/SA model to
treat the solvent as a fully equilibrated analytical continuum start-
ing the near the van der Walls surface of the solute. The parameters
were set according to the following: the energy window for saving
structures was 50 kJ/mol; the maximum iterations for minimizing
the molecules were 5000; the convergence threshold was 0.05; and
the others adopted the default values.
It has been well documented that phosphotyrosine is important
for linear peptide ligand binding to Grb2-SH2 domain by forming
extensive hydrogen bonds and hydrophobic interactions.6,33 Delet-
ing unnecessary amino acids of G1TE, and phosphorylation of Tyr3
of G1TE provided for us peptide 1 that competitively bound to the
Grb2-SH2 domain protein with an Kd of 3.6 lM. The inhibitory
activity of pTyr-containing cyclic pentapeptide 1 is at least 7 times
more potent than the corresponding G1TE. Molecular modeling
indicates that the negatively charged pTyr oxygens of cyclic penta-
peptide 1 form key binding interactions with the Arg 67 and Arg 86
and the three Ser residues 88, 90 and 96 of the protein, and consen-
sus amino acid Glu 2 of the cyclic pentapeptide 1 form two extra
hydrogen bonds with residue Arg 67 because of the strong posi-
tive-negative charges attraction (Fig. 4A).
The X-ray structure of Grb2-SH2 domain complexed with pTyr-
containing peptide ligand has shown that the phosphopeptide
binds to Grb2-SH2 domain in a b-turn conformation due to the
existence of Trp121 around the asparagine binding pocket.6 There-
fore, we intentionally introduced a turn-inducing amino acid, such
as 1-amino-1-cyclohexylic acid (Ach), in the position 2. When
replacing Glu 4 with Ach 4, the resulting peptide 2 loses charge

2696
S. Jiang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2693–2698
Figure 4. Docking poses of cyclic peptides 1 (A), 2 (B), 15 (C, R enantiomer) and 16 (D) with Grb2-SH2 domain (PDB ID 1TZE). Hydrogen bonds between ligands and protein
are represented by yellow dot lines. For clarity, only polar hydrogen atoms are shown here.
interactions with Arg 67, but gains hydrophobic Van der Waals
interactions with Gln 106 and Phe 108 of the Grb2-SH2 domain,
as shown in Figure 4B. In addition, the 310 helix turn, with the tor-
vored change of the peptide backbone conformation. This trend
was also observed in the Tyr 4, Trp 4, and Nal 4-containing pep-
tides 3, 4, 5, 8, 9, 10, 11 and 12. Compared to thioether-bridged
analogues, the activities of these sulfoxide bridged cyclopeptides
are not improved, due to the steric hindrance caused by the bulky
side chain of tyrosine, tryptophan, and 2-naphthylalanine, respec-
tively. We also incorporated Polyamide linkage into cyclic penta-
peptides 2, and examined the potency to determine the optimal
linkage which favors forming the b-turn like conformation. The
sion angles u = À62.1o,
w
= À34.3o and
x
= 170.4o, induced by Ach
is also favored for the peptide binding to the Grb2-SH2 domain.
The overall effect makes peptide 2 (Kd = 1.42
2.5-fold more potent than peptide 1.
lM) approximately
By analyzing the model of the complex structure of G1TE with
Grb2-SH2 domain, we found that Val 4 has van der Waals interac-
tions with the side chains of Lys 109 and Leu 120 of the Grb2-SH2
domain. In addition, Val 4 localizes in a hydrophobic region of the
Grb2 protein, which is composed of Phe 108, Gln 106, Trp 121, Lys
109, and Leu 120. Therefore, introducing a more hydrophobic ami-
no acid residue into position 4 of cyclic pentapeptide 2 would im-
prove this kind of van der Waals interactions. Considering these
characteristic interactions, we incorporated a series of hydropho-
bic amino acids in position 4, as shown in Table 1. The binding
binding affinity of the resulting peptide 16 (Kd = 7.55 lM) was
5.3 times less than its precursor thioether analog 2. This indicates
that the rigid cyclization constraint conferred by the backbone
cyclization disfavors either the formation of b-turn conformation
or the optimal orientation of the side chains of essential residues.
These results indicate that the linkage in cyclic pentapeptides,
which are not involved in the direct interactions with the binding
pocket of the protein, still has a function to induce the required
conformation via its backbone.
affinity of the modified peptides
3 (Kd = 1.06 lM) and 4
(Kd = 1.05 M) increases approximately 1.5-fold when replacing
l
4-Amino-1,1-dioxo-hexahydro-thiopyran-4-carboxylic acid (Atp)
and 3-amino-piperidine-4-carboxylic acid (Acp) are turn-inducing
amino acids, similar to Ach. We replaced Ach 2 with Atp and Acp,
respectively, whereby the resulting cyclic peptides 14 and 15 showed
1.6 and 4 times more inhibitory activity than the corresponding
Val 4 with tyrosine (Tyr) or tryptophan (Trp). Compared to valine,
these amino acids are more hydrophobic, which results in favor-
able and well defined Van der Waals interactions. However, these
improved interactions can be overweighed by increasing exposed
hydrophobic surface area under some circumstances. For instance,
unmodified peptide 2, respectively (14, Kd = 0.88
lM; 15, Kd =
the binding affinity of the resulting peptides 5 (Kd = 2.71
lM) was
0.359 M). Molecular modeling shows that that the replacing would
l
not improved, and even decreased when Val 4 was substituted
with L-2-naphthylalanine (2-Nal).
induce more hydrogen-bond interactions with Leu 120 and Lys 109 of
the Grb2-SH2 domain (Fig. 4C).
To further constrain the backbone conformation of cyclic penta-
peptides, we also tried to modify the thioether bridge of these pep-
tides, since this –CH2–S–CH2– motif is conformational flexible and
may contribute to an entropic penalty for the thioether-bridged
cyclic pentapeptides binding to Grb2 protein. When the thioe-
ther-cyclized peptide 2 was oxidized into the relatively more rigid
R-configured sulfoxide 6 and S-configured sulfoxide 7, the binding
There are two intramolecular hydrogen bonds, which are formed
by a same oxygen atom with two same hydrogen atoms in the 18
member ring, in almost all these 16 compounds’ docking conforma-
tions (Fig. 5E is as an example), except that cyclic peptide 1 has three
(Fig. 5B). This extra hydrogen bond is created by the propanoic acid
group with the amide group sitting next to it. But for the global min-
imum conformations we searched, the situations are different. Cyc-
lic peptide 15, actually, is a racemic mixture. For its R enantiomer,
the global minimum conformation has only one intramolecular
affinity was slightly decreased (6, Kd = 1.56
lM; 7, Kd = 1.76 lM)
than its precursor thioether analog 2, probably due to the disfa-

S. Jiang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2693–2698
2697
Figure 5. Conformation analyses of compounds 1, 15 and 16. Intramolecular hydrogen bonds are represented by yellow dot lines. For clarity, only polar hydrogen atoms are
shown here.
hydrogen bond, produced by the phosphoric acid group and the
charged amino atom in the rigid six-member ring (Fig. 5C). Whereas,
its S enantiomer’s global minimum conformation has four hydrogen
bonds(Fig. 5D), whichmakesit muchmoredifficulttochangeitsglo-
balminimumconformationtobindingconformationthanR enantio-
mer. So we can conclude that R enantiomer of cyclic peptide 15 has
stronger binding affinity than S enantiomer just from conforma-
tional point. Cyclic peptide 1 can even produce six intramolecular
hydrogen bonds (Fig. 5A), among which three are through the flexi-
ble propanoic acid group; Cyclic peptide 16 can produce four
(Fig. 5F), one of which is formed by the negative charged phosphoric
acid group with positive charged amino group and therefore is much
stronger than other hydrogen bonds. So, for the same reason, for cyc-
lic peptides 1 and 16, it is also much harder to change their global
minimum conformations to binding conformations than other ana-
logues. This, from the other side, explains why cyclic peptide 15 has
best and cyclic peptides 1 and 16 have worst biological activities to-
ward Grb2-SH2 domain.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.134.
References and notes
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with a Kd = 0.359
lM, as evaluated by Biacore assays. This fully
elaborated small peptidomimetic provides us a novel template
for the development of pharmacokinetically improved peptidomi-
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as potential therapeutic agents for the treatment of erbB2-related
cancers.
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Acknowledgments
This project was supported by the National Natural Science Foun-
dation (Grant no. 20802078), National Basic Research Program of
China (973 Program, Grant no. 2009CB940900), the Guangdong
Natural Science Foundation (Grant no. 8151066302000008) and
the Intramural Research Program of the NIH, National Cancer Insti-
tute, Center for Cancer Research.
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