Development of non-peptide ligands of growth factor receptor-bound protein 2-src homology 2 domain using molecular modeling and NMR spectroscopy

Article abstract of DOI:10.1021/jm101478n

We report a novel series of non-peptide ligands that inhibit the growth factor receptor-bound protein 2 (Grb2)-Src homology 2 (SH2) domain binding, designed using a combined computational and NMR-driven approach. We have identified a new lead compound, 1n (IC₅₀ = 56 μM), which is cytotoxic in HER2-positive breast cancer cells and disrupts the interaction between HER2 and Grb2. Thus, 1n can be used as a scaffold for the development of efficient Grb2-SH2 domain binding inhibitors.

Full text of DOI:10.1021/jm101478n

1096 J. Med. Chem. 2011, 54, 1096–1100
DOI: 10.1021/jm101478n
Development of Non-Peptide Ligands of Growth Factor Receptor-Bound Protein 2-Src Homology 2
Domain Using Molecular Modeling and NMR Spectroscopy†
‡
Angel L. Orcajo-Rincon, Silvia Ortega-Gutierrez, Pedro Serrano, Ivan R. Torrecillas, Kurt Wuthrich,
‡
§
^
§,
ꢀ
ꢀ
ꢀ
Mercedes Campillo, Leonardo Pardo, Alma Viso, Bellinda Benhamu, and Marı
€
a L. Lopez-Rodrı
´
´
guez*,‡
^
^
‡
ꢀ ‡
ꢀ
‡
´
ꢀ
´
Departamento de Quımica Organica I, Facultad de Ciencias Quımicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain,
§Department of Molecular Biology, and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
92037 La Jolla, California, United States, and ^Laboratori de Medicina Computacional, Unitat de Bioestadistica, Facultat de Medicina,
Universitat Autonoma de Barcelona, E-08913 Bellaterra, Barcelona, Spain
Received November 17, 2010
We report a novel series of non-peptide ligands that inhibit the growth factor receptor-bound protein 2
(Grb2)-Src homology 2 (SH2) domain binding, designed using a combined computational and NMR-
driven approach. We have identified a new lead compound, 1n (IC50=56 μM), which is cytotoxic in
HER2-positive breast cancer cells and disrupts the interaction between HER2 and Grb2. Thus, 1n can
be used as a scaffold for the development of efficient Grb2-SH2 domain binding inhibitors.
Introduction
Grb2-SH2 domain binding inhibitors as tools for validation
of anticancer therapies based on the blockage of the Grb2-
mediated downstream pathways.
The human epidermal growth factor receptor 2 (HER2a or
ErbB-2) is an important therapeutic target in breast cancer,
which is overexpressed in around 25% of the invasive breast
cancers and associated with poor disease-free survival and
resistance to chemotherapeutic drugs.1-3 Blockage of HER2
activation by inhibiting its interaction with the Src homology
2 (SH2) domainofthe growth factor receptor-bound protein2
(Grb2) has emerged as a new drug discovery strategy to
identify novel therapeutic agents that selectively block the
HER2 signaling pathway.4 Oligopeptides have been reported
as strong inhibitors of the HER2-Grb2 interaction by block-
ing the Grb2-SH2 domain.5 However, features such as limited
stability or bioavailability have compromised their use in vivo
and subsequently the definitive validation of Grb2-SH2 do-
main binding inhibition as a useful therapeutic approach.
In this regard and in terms of finding non-phosphopeptide
compounds as Grb2-SH2 ligands, very little progress has been
reported.6,7 Therefore, the development of new non-peptide
small molecules capable of disrupting binding between HER2
and Grb2 might bolster research in this field and overcome the
limitations of the currently available inhibitors.
In this paper we describe a structurally novel series of small
molecules that bind to the Grb2-SH2 domain. These com-
pounds have been designed using molecular modeling techni-
ques and evaluated by an ELISA-type assay. NMR chemical
shift perturbation studies with the ligand 1k enabled the
identification of its binding site in the SH2 domain of Grb2
and to confirm that this family of molecules reproduces the key
interactions predicted by the computational models. These
results allowed the design of lead compound 1n (IC50=56 μM),
which represents a new scaffold to develop more efficient
Results and Discussion
Inthe searchforGrb2-SH2ligandswithastructurally novel
core, we first identified the residues involved in key interac-
tions in the crystal structure of the Grb2-SH2 domain in com-
plex with the high-affinity 2-Abz-EpYINQ-NH2 pentapep-
tide.8 This structure reveals the pivotal roles of two protein
sites in the interaction with the inhibitor. The first site is a
hydrophilic pocket highly conserved among SH2 domains,
which is responsible for pTyr binding and is defined by a
cluster of serine residues, Ser88 (βB7), Ser90 (βC2), and Ser96
(βC3) (the positions in parentheses indicate the topology of
the Grb2-SH2 domain, following the commonly used notation
of Eck and collaborators9), and the positively charged amino
acids Arg67 (RA2) and Arg86 (RB5) (Figure 1A). The residue
Arg67 (RA2) is especially important, since it is involved in the
interactions with the N-terminal domain of the peptide and
forms a hydrogen bond with the backbone carbonyl group of
the pY - 1 amino acid. The second site includes the backbone
atoms of the amino acids His107 (βD4), Lys109 (βD6),
and Leu120 (βE4), which form four hydrogen bonds with the
pY þ 1 and pY þ 2 positions of the inhibitor (Figure 1A).
According to these interactions, we designed non-peptide com-
pounds with a novel structural core (Table 1): (i) the W
substituent mimics the backbone carbonyl group of the amino
acid in position pY - 1 that interacts with Arg67 (RA2); (ii) the
pTyr is linked to the spacer by an amide group, which preserves
the hydrogen bond with the backbone carbonyl group of His107
(βD4) observed for the N-H group of the pY þ 1 residue in
2-Abz-EpYINQ-NH2; (iii) the terminal group is designed to
contact the backbone carbonyl and the N-H groups of Lys109
(βD6), mimicking the role of Asn in position pY þ 2
(Figure 1B); (iv) spacers of two and three methylene units were
chosen to provide enough distance and flexibility to enable
concerted interactions with the two binding sites.
†Dedicated to Professor Antonio Garcı
his retirement.
*To whom correspondence should be addressed. Phone: þ34
913944239. Fax: þ34 913944103. E-mail: mluzlr@quim.ucm.es.
a Abbreviations: Grb2, growth factor receptor-bound protein 2; HER2,
human epidermal growth factor receptor 2; SH2, Src homology 2.
a Martınez on the occasion of
´ ´
r
pubs.acs.org/jmc
Published on Web 01/27/2011
2011 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1097
Figure 1. Representative structures obtained in the molecular dynamics (MD) simulations of Grb2-SH2 in complex with (A) the 2-Abz-EpYINQ-
NH2 pentapeptide (PDB code 1ZFP) and with (B) 1k and (C) 1n. (D-G) Evolution of dihedral angles χ1 (black) and χ2 (gray) in the vicinity of the
cyclohexyl ring of 1n (E, G) and the analogous angles in 1k (D, F) obtained during the MD simulations of the ligands in bulk water. Representative
structures of 1k (D) and 1n (E) obtained during the simulations in water are shown. (H) Binding free energy decomposition on a per residue basis
for Grb2-SH2 in complexes with 2-Abz-EpYINQ-NH2 (blue), 1k (red), and 1n (green).
The interactions between the Grb2-SH2 domain and 1k
relative to the 2-Abz-EpYINQ-NH2 pentapeptide were eval-
uated by comparing molecular dynamics (MD) simulations
for the two complexes performed with MM-GBSA methodo-
logy.10 In both cases equilibration was achieved during the
first 5 ns of simulation, as monitored by the all-atoms root-
mean-square deviation (rmsd) and from key interatomic ionic
and hydrogen bond distances between Grb2-SH2 and either
2-Abz-EpYINQ-NH2 or 1k as a function of time. It is worth
mentioning that for 1k (Figure 1B) and all other designed
molecules (data not shown) the key interactions with Arg67
(RA2), His107 (βD4), and Lys109 (βD6) were maintained
during the full simulation time, similar to what was observed
for 2-Abz-EpYINQ-NH2 (Figure 1A).
The synthesis of 1a-j was accomplished by phosphoryla-
tion ofcommercial N-acetyl- or N-benzoyl-L-tyrosine or ofthe
readily available N-butyryl-L-tyrosine 2, which was followed by
condensation with the appropriate amine and subsequent de-
protection of the phosphate group (Scheme 1). Intermediates
3a-c were prepared by a one-pot procedure, which involved the
temporary protection of the carboxyl group of N-acyl-L-tyrosine
as a silyl ester followed by in situ phosphitylation of the tyrosyl
hydroxyl group and oxidation of the resultant phosphite triester.
Derivatives 4a-j were obtained by condensation of 3a-c with
the corresponding amine in the presence of EDC as a coupling
reagent and HOBt as an additive to minimize racemization.11
Final removal of the benzyl groups by catalytic hydrogenation
of protected amides 4a-j yielded the target compounds 1a-j.
Noncommercial amines 1-(3-aminopropyl)tetrahydropyrimi-
din-2(1H)-one (5a) and 1-(2-aminoethyl)imidazolin-2-one (5b)
were obtained by treatment of N-(3-aminopropyl)propane-1,
3-diamine or N-(2-aminoethyl)ethane-1,2-diamine, respectively,
with urea. The amount of racemization following this synthetic
protocol was estimated by coupling N-acetyl-L-tyrosine with
(1S)-1-phenylethylamine and comparing the area of the peaks
of methyl groups of 1-phenylethylamine in the 1H NMR spec-
trum. The ratio between the two diastereomers [S/S (1.38 ppm);
S/R (1.49 ppm)] was 5:1 (data not shown). Therefore, we
decided to synthesize the enantiopure isomers of the most potent
compound in the series (1a; see Table 1), i.e., 1k and 1l, which
were obtained using the 9-fluorenylmethyloxycarbonyl (Fmoc)
protecting group (Scheme 2) to prevent racemization. After
phosphorylation, Fmoc derivative 6a or 6b was condensed with
amine 5a, following the above-mentioned procedure. The acetyl
group was introduced after Fmoc removal, and amides 8a,b
were finally debenzylated to yield the (R)- and (S)-N-acetyl-
N0-[3-(2-oxoperhydropyrimidin-1-yl)propyl]-O-phosphonotyr-
osinamides 1l and 1k (Scheme 2). Racemic 1m was obtained
from N-acetyl-D,L-tyrosinefollowingtheprocedureinScheme1.
The ability of the synthetic compounds to bind to the Grb2-
SH2 domain was determined by an ELISA competition test
using Grb2-SH2 and the phosphopeptide biotin-Ahx-PSpYV-
NVQN12 (Table 1). Among this series of ligands, 1k showed the
highest affinity and was selected to study its binding to Grb2-
SH2 by NMR chemical shift perturbation experiments. The
data were recorded using the 98-residue Grb2-SH2 construct
described by Waugh and colleagues, which comprises the poly-
peptide segment 55-153,13 and the assignments of the back-
bone amide resonances were taken from the work by Thanabal
and colleagues.14 The resonance assignments of Grb2-SH2
in complex with 1k were obtained by analyzing a series of
[15N,1H]HSQC spectra of samples containing 15N-labeled

1098 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Table 1. Affinity of Compounds 1a-n for the Grb2-SH2 Domain
Orcajo-Rincoꢀn et al.
significant chemical shift differences, which are indicative of
structural rearrangements upon binding of 1k (Figure 2B). The
most strongly affected residue in region I is Arg67 (RA2), which
interacts with the N-terminal carbonyl and phosphate groups
of 1k, similar to previous observations with different li-
gands.7,15-17 Sequence regions II and III correspond to a hydro-
philic, positively charged surface area involved in the stabiliza-
tion of the phosphate group by means of electrostatic and
hydrogen bond interactions. The chemical shift differences
observed for Arg86 (βB5), Ser88 (βB7), and Ser96 (βC3)
are particularly relevant, since these residues have previously
been reported to be fundamental for the interaction with
pTyr.13,15-17 In regions IV and V, large chemical shift differ-
ences are seen for His107 (βD4) and Lys109 (βD6), which are
engaged in hydrogen bond interactions with 1k through their
backbone amide groups. The neighboring residues Phe108,
Val110, Leu111, and Trp121 (EF1) form a lipophilic pocket
that stabilizes the protein-inhibitor complex by van der Waals
interactions with the terminal group of 1k.15,16 The strong effect
on Trp121 (EF1) is of special interest, since this residue is
responsible for the specificity of ligand binding to Grb2 when
compared to other SH2 domain-containing proteins.7,15-17
Overall these results indicate that the residues with significant
chemical shift perturbations due to the presence of bound 1k
(Figure 2C) coincide with those found to form a binding pocket
in several crystal or solution structures of Grb2-SH27,15-17 and
validate the assumptions used to design this new family of Grb2-
SH2 binding blockers. Hence, we attributed the moderate
affinity of 1k to a high degree of conformational freedom due
to the methylene spacer so that improved binding might be
achieved by introducing conformational restrictions in this part
of the molecule. Thus, a new compound, 1n, was designed using
1k as a scaffold and introducing in the spacer a conformation-
ally constrained cyclohexyl ring. MD simulations in an explicit
water environment with 1k and 1n were performed to evaluate
the rigidity introduced by the cyclohexyl group in terms of the χ1
and χ2 angles (Figure 1D-G). Molecule 1k is highly flexible,
adopting different conformations during the simulation time,
while the cyclohexyl ring of 1n constrains the backbone, thus
favoring the β-turn conformation required for Grb2 binding
and the interactions with Phe108 (βD5) and Trp121 (EF1)
(Figure 1C). The stability of 1n in complex with Grb2-SH2 was
also evaluated using the MM-GBSA methodology10 and com-
pared with the results obtained previously for the pentapeptide
2-Abz-EpYINQ-NH2 and 1k. The binding free energy decom-
position on a per residue basis is shown for the three complexes
in Figure 1H, where 1k and 1n exhibit similar interaction
profiles as 2-Abz-EpYINQ-NH2, with the only exceptions of
Ser90 (βC2) and Leu120 (βE4). The absence of the anthranyl
moiety of the peptide in 1k and 1n seems to facilitate the
interaction between Ser90 and pTyr, whereas the terminal
groups of 1k and 1n cannot interact with the backbone amide
of Leu120 (βE4) (see Figure 1).
a Competition binding assays were conducted as described in the
Supporting Information. Dose-response relationships were generated
by nonlinear regression fits of the competition curves with the software
Prism. Data are expressed as the mean ( SEM of at least three indepen-
dent experiments carried out in duplicate. b For N2-{[1-({(2R)-2-carboxy-
methyl-3-[4-phosphonomethyl)phenyl]propanoyl}amino)cyclohexyl]car-
bonyl}-N1-[3-(1-naphthyl)propyl]-L-aspartamide (122A-130, 9), kindly
donated by Prof. T. R. Burke, which was used as a control of the ELISA
test, we obtainedIC50 values comparable to those previously reported (see
8a, ref 21). c Inactive compounds displace less than10% of the phosphopeptide
at the maximal concentration used (5 mM).
Grb2-SH2 and different ratios of compound 1k (5:1 f 1:5).
Figure 2A shows a superposition of two 2D [15N,1H]HSQC
spectra of Grb2-SH2 acquired in the absence and presence (1:1
molar protein/compound ratio) of 1k, from which the residues
with large chemical shift changes were identified. As a control,
to rule out that these shifts arise solely from interactions due to
the presence of pTyr, which is a common moiety in all the Grb2-
SH2 inhibitors reported so far, [15N,1H]HSQC spectra of the
protein in the presence of a 2-fold excess of N-acetylphospho-
tyrosine were obtained under identical conditions as the spectra
of Figure 2A. It was found that this compound induces chemical
shifts exclusively in a small number of Grb2 amino acid residues,
which are all located in the pTyr binding pocket (Supporting
To evaluate the biological effect of the cyclohexyl group in
the spacer, 1n was prepared following the procedure indicated
in Scheme 2 starting from L-tyrosine and 1-[2-(1-aminocyclo-
hexyl)ethyl]tetrahydropyrimidin-2(1H)-one (5c). The affinity
of 1n for Grb2-SH2 was found to be 3-fold higher than for 1k,
inhibiting Grb2-SH2 domain binding with an IC50 of 56 μM
(Table 1) in a competitive manner (Supporting Information
Figure S2). We further assessed the in vitro potential of the
new inhibitor in a HER2-positive MCF7 human breast cancer
cell line. 1n inhibited proliferation of these cells with an IC50 of
100 μM. In addition, at this concentration, no statistically
1
Information Figure S1). A plot of the combined H and 15N
chemical shifts for each residue, Δδav, versus the Grb2-SH2
amino acid sequence shows five regions, I-V, that undergo

Brief Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1099
Scheme 1. Synthesis of Compounds 1a-ja
a Reagents: (a) (i) NMM, tBuMe2SiCl, CH3CN; (ii) 1H-tetrazole, iPr2NP(OBn)2; (iii) tBuOOH (aq); (b) (i) HOBt, EDC, CH2Cl2, R-NH2; (c) H2/
Pd(C)/EtOH.
Scheme 2. Synthesis of Enantiopure Derivatives 1k, 1l, and 1na
a Reagents: (a) (i) NMM, tBuMe2SiCl, CH3CN; (ii) 1H-tetrazole,
iPr2NP(OBn)2; (iii) tBuOOH (aq); (b) HOBt, EDC, CH2Cl2, 5a or HOBt,
DIC, THF, 5c; (c) Et2NH, AcCl, CH3CN; (d) H2/Pd(C)/EtOH.
significant effect was observed on fibroblasts, ruling out a
general cytotoxicity of the compound (Figure 3A). To further
confirm the involvement of HER2-Grb2 interaction in these
effects, we assessed the cytotoxicity of 1n in two different
HER2-negative breast cancer cells, HER2-negative MCF7
and MDA-231 cell lines. In both cases, 1n did not show any
significant effect (Figure 3B). In addition, we studied the
direct interaction between HER2 and Grb2 by immunopre-
cipitation experiments. HER2-positive MCF7 cells were
treated with 1n or vehicle, washed, and lysed. HER2 was
immunoprecipitated and HER2 and Grb2 immunoblotted
using specific antibodies (Figure 3C), and the bands were
densitometered. Our results showed a 30% decrease of the
interaction between HER2 and Grb2 in the presence of 1n.
Taken together, theseresults clearly support that the cytotoxic
effect of 1n can be at least partially ascribed to its binding to
the Grb2-SH2 domain and the concomitant inhibition of the
HER2-Grb2 interaction. It is conceivable that interaction of
the ligand with residue Trp121 (EF1, responsible for the
specificity of ligand binding to Grb2 vs other SH2-containing
proteins7,15-17) could confer some degree of selectivity for the
recognition of Grb2-SH2 domain.
In summary, using a computation- and NMR-guided
Figure 2. NMR chemical shift mapping studies. (A) 2D [15N,1H]-
HSQC spectra of the Grb2-SH2 domain free (blue) and in complex with
1k (red) (molar ratio protein/compound 1:1). (B) Plot of the amino acid
sequence vs the chemical shift changes in the 1H-15N backbone moieties
of the Grb2-SH2 domain due to 1k binding. (C) Stereoribbon presenta-
tion of the Grb2-SH2 domain. The side chains of the residues that exhi-
bit chemical shift differences larger than 0.1 ppm (B) are identified.
approach, we designed a structurally novel non-peptide in-
hibitor of the Grb2-SH2 binding, 1n, which binds to the Grb2-
SH2 domain with an IC50 of 56 μM. This compound selec-
tively inhibits proliferation of the HER2-positive MCF7
breast cancer cell line with no apparent toxicity toward
fibroblasts and no effect on two different HER2- negative
breast cancer cell lines. Furthermore, 1n is able to disrupt the
interaction HER2-Grb2. Therefore, this compound is a pro-
mising scaffold for the development of efficient Grb2-SH2
domain binding inhibitors able to block the Grb2-associated
proliferative downstream pathways.

1100 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Orcajo-Rincoꢀn et al.
superposition of the [15N,1H]HSQC spectra of Grb2-SH2 in the
presence and absence of N-acetylphosphotyrosine; competition
binding curves for 9 and 1n. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Experimental Section
Full synthetic details and characterization data, including ele-
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compounds tested are >95% pure by elemental analysis. N-Butyryl-
L-tyrosine (2),18 N-acetyl-O-[bis(benzoyloxy)phosphoryl]-L-tyrosine
(3a),19 and amines 5a20 and 5b20 were synthesized according to
previously described procedures. Detailed synthetic procedures
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described in the Supporting Information.
Synthesis of N-Acyl-O-phosphonotyrosinamides (1a-n). To a
solution of the corresponding amides 4a-j, 8a-c (1 equiv) in
absolute EtOH (10 mL), 10% Pd(C) was added (200 mg), and
the suspension was hydrogenated at room temperature at an
initial pressure of 45 psi until the disappearance of the starting
benzylated amide (TLC). The mixture was filtered over Celite
and the solvent was removed under reduced pressure to yield the
pure debenzylated amides 1a-n (34-93%), which were then
recrystallized from the appropriate solvent(s).
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Acknowledgment. This work was supported by grants from
MICINN (SAF-2007/67008-C02 and SAF-2010/22198-C02),
CAM (S-SAL-249-2006) and ISC (RD07/0067). The authors
thank CAM for a predoctoral fellowship to A.L.O.-R. and
MICINN and the European Social Fund for a postdoctoral
fellowship to P.S. and for a Ramon y Cajal grant to S.O.-G. K.W.
is the Cecil H. and Ida M. Green Professor of Structural
Biology at The Scripps Research Institute. We thank Prof.
T.R. Burke, Jr. for the gift of compound 9, Prof. D.S. Waugh and
Prof. I. Brautin for providing us with the Grb2-SH2 plasmids,
and Dr. T. Puig and Dr. R. Colomer for donating the cell lines.
Supporting Information Available: Synthetic procedures for
the intermediates 3a-c, 4a-j, 5c, 6a,b, 7a-c, and 8a-c; analy-
tical and spectral characterization data of the synthesized
compounds; experimental details for determination of bind-
ing affinity, protein sample preparation, NMR methods, in
vitro cell cytotoxicity, and immunoprecipitation experiments;
(20) Hurwitz, M. D.; Auten, J. P. US2613212, 1952.
(21) Wei, C.-Q.; Li, B.; Guo, R.; Yang, D.; Burke, T. R., Jr. Develop-
ment of a phosphatase-stable phosphotyrosyl mimetic suitably
protected for the synthesis of high-affinity Grb2 SH2 domain-
binding ligands. Bioorg. Med. Chem. Lett. 2002, 12, 2781–2784.