Synthesis and structural characterization of a monocarboxylic inhibitor for GRB2 SH2 domain

Article abstract of DOI:10.1016/j.bmcl.2021.128354

A monocarboxylic inhibitor was designed and synthesized to disrupt the protein–protein interaction (PPI) between GRB2 and phosphotyrosine-containing proteins. Biochemical characterizations show compound 7 binds with the Src homology 2 (SH2) domain of GRB2 and is more potent than EGFR₁₀₆₈ phosphopeptide 14-mer. X-ray crystallographic studies demonstrate compound 7 occupies the GRB2 binding site for phosphotyrosine-containing sequences and reveal key structural features for GRB2–inhibitor binding. This compound with a –1 formal charge offers a new direction for structural optimization to generate cell-permeable inhibitors for this key protein target of the aberrant Ras-MAPK signaling cascade.

Full text of DOI:10.1016/j.bmcl.2021.128354

Bioorg. Med. Chem. Lett. 51 (2021) 128354
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structural characterization of a monocarboxylic inhibitor for
GRB2 SH2 domain
,
,
,
,*
Tao Xiaoa 1, Luxin Suna 1, Min Zhanga, Zilu Lia c, Eric B. Haurab, Ernst Schonbrunna
,
,c,*
Haitao Jia
a Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, United States
b Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, United States
c Department of Chemistry, University of South Florida, Tampa, FL, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
A monocarboxylic inhibitor was designed and synthesized to disrupt the protein–protein interaction (PPI) be-
tween GRB2 and phosphotyrosine-containing proteins. Biochemical characterizations show compound 7 binds
with the Src homology 2 (SH2) domain of GRB2 and is more potent than EGFR1068 phosphopeptide 14-mer. X-ray
crystallographic studies demonstrate compound 7 occupies the GRB2 binding site for phosphotyrosine-
containing sequences and reveal key structural features for GRB2–inhibitor binding. This compound with a –1
formal charge offers a new direction for structural optimization to generate cell-permeable inhibitors for this key
protein target of the aberrant Ras-MAPK signaling cascade.
GRB2
Protein-Protein Interaction
SH2 domains
Inhibitors
Macrocyclic peptides
X-ray crystallography
Ras-MAPK signaling
Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein
that mediates activation of mitogenic Ras signaling pathways.1–2 After
growth factor stimulation, phosphotyrosine (pTyr)-containing se-
quences of active receptor tyrosine kinases (RTKs) bind to the central Src
homology 2 (SH2) domain of GRB2, and the N- and C-terminal SH3
domains of GRB2 bring the nucleotide exchange factor SOS to the cell
membrane to activate the Ras–mitogen-activated protein kinase (MAPK)
signaling cascade for cell growth and differentiation. Aberrant GRB2-
dependent Ras activation significantly contributes to cancer develop-
ment and progression.1–3 The SH2 domain of GRB2 can also bind with
pTyr-containing sequences of adaptor proteins, such as the Src homol-
ogous and collagen (SHC) protein, and non-receptor tyrosine kinases,
such as BCR-ABL, to mediate aberrant Ras–MAPK signaling.4 Hence,
GRB2 is a key, convergent MAPK signaling node and an interesting
protein target for anticancer drug development.
inhibitors that bind to GRB2 SH2 domain and disrupt the protein–pro-
tein interaction (PPI) between GRB2 and pYXNX-containing
proteins.10–14 Some interesting compounds are listed in Fig. 1. Com-
pound 1 was extracted from an EGFR pTyr-containing sequence. This
compound inhibited EGFR/GRB2 PPI with a half maximal inhibitory
concentration (IC50) of 8.6
μ
M.15 Compound 2 was evolved from 1 and
exhibited an IC50 two orders of magnitude lower than 1.16 The cycli-
zation of 2 to mimic the β-turn binding conformation of pTyr-containing
sequences with GRB2 led to 3.17 The introduction of a carboxymethyl
group to 3 offered 4 with an IC50 of 2 nM.18 The crystal and NMR
structures of GRB2 SH2 domain in complexes with these inhibitors were
reported.5–6,19–30 However, despite excellent in vitro biochemical ac-
tivities, these compounds showed overall poor cellular activities,
although cell-based data were collected with some cell lines.17–18,31–34
The phosphate and phosphonomethyl groups in these compounds car-
rying –2 charges at physiological pH were thought to cause poor cell
permeability.9–11,35–36 Phosphate mimicking bioisosteres were intro-
duced to 2–4, and compounds 5 and 6 were reported as potent GRB2
inhibitors in biochemical assays.31,37–43Again, studies indicated 5 and 6
displayed unsatisfying cell-based and in vivo activities for further
development.31,38,40–41,44–47 The latter medicinal chemistry studies
were switched to understand molecular recognition questions using
X-ray crystallographic5–6 and NMR7 studies indicated that pTyr-
containing sequences pYXNX (pY, X, and N represent phosphotyr-
osine, any residue, and asparagine, respectively) adopt a type I β-turn
conformation to bind to GRB2 SH2 domain. This conformation is
different from the extended conformation of pTyr-containing sequences
observed in many other SH2 domain-containing proteins.8–9 Highly
appreciable medicinal chemistry efforts have been made to discover
* Corresponding authors.
E-mail addresses: Ernst.Schonbrunn@moffitt.org (E. Schonbrunn), Haitao.Ji@moffitt.org (H. Ji).
1
Contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2021.128354
Received 13 July 2021; Received in revised form 29 August 2021; Accepted 2 September 2021
Available online 7 September 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

T. Xiao et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128354
GRB2 SH2 domain and inhibitors as a model system, and important
knowledge was obtained about ligand conformational constraints,
fluorescein (FITC)-labelled human EGFR1068 phosphopeptide, FITC-
Ahx-PVPEpYINQSVPKRK-NH2, (Ahx: 6-aminohexanoic acid) was syn-
thesized and purified (HPLC purity > 95%). The dissociation constant
(KD) of the EGFR/GRB2 PPI from fluorescence anisotropy binding ex-
periments was around 191 nM, which is consistent with reported KD, as
shown in Fig. 2A.54–55 The fluorescence anisotropy competitive inhibi-
tion assay was then used to evaluate the inhibitory activities of 3, 7 and
8.
nonpolar surface area burial, and cation-
π
interaction.27–30,48–49
We decided to substitute the phosphonomethyl group in 3 with a
carboxylic acid group and designed compound 7 in Fig. 1B to examine
the binding affinity difference between 3, 7, and EGFR1068-containing
phosphotyrosyl peptide. Out of all EGFR sequences, the EGFR1068-con-
taining phosphopeptide is known to have the highest binding affinity
with GRB2. The methyl ester 8 in Fig. 1B was also synthesized for
comparison.
As shown in Fig. 2B, the inhibition constant (Ki) of 7 for disruption of
the EGFR/GRB2 PPI is 140 nM. This compound was 2-fold more potent
than the EGFR1068 phosphopeptide 14-mer that displayed a Ki of 400 nM
in the parallel assays, offering an exciting starting point for further in-
hibitor optimization. It is noted that the positive control, compound 3,
displayed a Ki of 16 nM. On the other hand, the Ki of the methyl ester of
7, compound 8, was > 20 µM, indicating the importance of the nega-
tively charged carboxylic acid group of 7 for the inhibitory activity.
Assessment of binding potential by differential scanning fluorimetry
(DSF) showed a significant increase in thermostability of GRB2 in the
To synthesize 7 and 8, the commercially available Evans’ chiral
auxiliary, (S)-(+)-4-phenyl-2-oxazolidinone, was first attempted to
prepare 12 in Scheme 1A. However, two diastereomers of 12 with a
diastereometric ratio (dr) of 3:1 were inseparable by column chroma-
tography. We then screened other Evans’ chiral auxiliaries, including
(S)-4-benzyl-2-oxazolidinone, (S)-(ꢀ )-4-isopropyl-2-oxazolidinone, (S)-
(ꢀ )-4-benzyl-5,5-dimethyl-2-oxazolidinone, and (S)-4-tert-butyl-2-oxa-
zolidinone. The chiral auxiliary (S)-4-tert-butyl-2-oxazolidinone (14)
offered the highest diastereomer selectivity for the desired product (dr
= 100:6). The synthetic route for 7 is shown in Scheme 1B. The reaction
between acrylic acid and acryloyl chloride gave acrylic anhydride 13,
which was used to acylate 14 under the LiCl and Et3N condition to afford
15 in 85% yield over two steps. The Heck reaction50 between 9 and 15
gave 16 in 76% yield, which underwent 1,4-addition with vinyl-
magnesium bromide under the PhSCu condition51 to afford 17 with
reproducible yield and high diastereoselectivity. The Evans’ chiral
auxiliary in 17 was removed by hydrolysis to give an acid, which was
then coupled with 18 to give 19. Key intermediate 18 was synthesized
by a route slightly modified based on that reported previously51, as
shown in Supplementary Scheme 1. Ruthenium-catalyzed ring-closing
metathesis of 19 using the second generation of Grubbs catalyst and
deprotection of the tert-butyl group under the acidic condition offered
the final product 7 as a single E isomer. The configuration of the alkene
was determined to be trans by 1H NMR which showed the coupling
constant (J) of 15 Hz for two vicinal alkene protons.
presence of compound 3 (ΔTm = 16.5 ◦C) and compound 7 (ΔTm
=
8.6 ◦C) (Fig. 2C). The data confirmed the differential binding potential
of 3 and 7 observed in fluorescence anisotropy assays (Fig. 2B).
Parallel artificial membrane permeability assays (PAMPAs) were
performed to assess the permeability of 3 and 7 through the artificial
membrane that was composed of 1% egg lecithin in n-dodecane, a useful
system to examine compound cell permeability. Compounds 3 and 7
(500 µM) were placed on the donor side of the membrane. After 5-h
incubation at room temperature, the amounts of 3 and 7 in the
receiving solution were quantified by HPLC analyses. The percent
transport (%T) and the apparent permeability coefficient (Papp) were
calculated using the previously reported equations56–57. As shown in
Table 1, the PAMPA results demonstrate that 7 displays good perme-
ability through the artificial membrane, while 3 has poor permeability
in this assay.
To gain structural insights in GRB2 inhibition, a crystal structure of
the GRB2 SH2 domain liganded with compound 7 was determined at
2.0 Å resolution (PDB code: 7MPH) (Fig. 3). The asymmetric unit is
composed of two GRB2 trimers, and each monomer interacts with one
molecule of 7 at full occupancy. The inhibitor establishes H-bonds with
the main chain atoms of Lys109 and Leu120 through the carboxamide
moiety, and with His107 through the N-cyclohexylacetamide moiety.
Additional H-bonds occur between the phenylacetate oxygens and the
side chains of Arg86 and Ser96, albeit not in all monomers, suggesting
weaker interaction potential. Side chains of other surrounding residues
stabilize the inhibitor through hydrophobic van der Waals interactions.
The naphthalene moiety is solvent exposed and presents a potential exit
Compound 8 was synthesized by esterification of 7 using CH3I under
the K2CO3/DMF condition. The positive control compound 3 was syn-
thesized by following the literature procedure (Supplementary Scheme
S2).51 The only difference was that (S)-4-tert-butyl-2-oxazolidinone was
used to increase the diastereoselectivity of the 1,4-addition reaction
with vinylmagnesium bromide.
Following the previously reported GRB2 fluorescence polarization
(FP) assay,52 we overexpressed N-terminally His6-tagged human full-
length GRB2 (residues 1–217) in E. coli and purified GRB2. It is known
that GRB2 is preferred to bind with pY1068 of EGFR.53 N-terminally
Fig. 1. (A) Reported GRB2 inhibitors. (B) Newly designed GRB2 inhibitors.
2

T. Xiao et al.
Bioorganic & Medicinal Chemistry Letters 51 (2021) 128354
Scheme 1. The synthetic route for compounds 7 and 8.
Fig. 2. (A) Fluorescence anisotropy binding experiments to determine the KD of full-length human GRB2 with a fluorescently labeled EGFR1068 phosphopeptide,
FITC-Ahx-PVPEpYINQSVPKRK-NH2. The data was expressed as mean ± standard deviation (n = 3). (B) fluorescence anisotropy competitive inhibition assays to
determine IC50s of 3, 7, 8, and EGFR1068 phosphopeptide for disruption of the interaction between full-length GRB2 and FITC-Ahx-PVPEpYINQSVPKRK-NH2. The Ki
values were derived. The data was expressed as mean ± standard deviation (n = 3). (C) Thermal stabilization of GRB2 SH2 domain by 3 and 7. The melting point (Tm)
of the SH2 domain was determined in the absence and presence of inhibitors by DSF.
missing in the structure with 7, offering the structural base why 7 is a
Table 1
weaker inhibitor than 3 in biochemical assays.
The PAMPA results of compounds 3 and 7.
In summary, we designed a new monocarboxylic inhibitor for GRB2
Compound
%T ± SD
Papp ± SD (cm⋅sꢀ 1, ×10–6
)
SH2 domain, compound 7 in Fig. 1B, and developed a robust synthetic
route to synthesize this class of macrocyclic peptides. We also developed
3
7
0
0
24.3 ± 2.5
19.2 ± 2.0
a
fluorescence anisotropy competitive inhibition assay to assess
disruption of the PPI between GRB2 and phosphotyrosine-containing
peptides. Compound 7 exhibits a Ki of 140 nM for disruption of EGFR/
GRB2 PPI and is 2-fold more potent than EGFR1068 phophopeptide 14-
mer. The crystallographic studies demonstrate compound 7 binds with
GRB2 SH2 domain and disclose key structural features for the non-
covalent binding of compound 7 with GRB2. This compound carrying a
–1 charge serves as an interesting lead compound for further optimiza-
tion to generate a novel targeted therapy for anticancer treatment.
vector for bivalent inhibitor design.
When compared to the crystal structures of GRB2 in complex with 6
(PDB IDs, 2AOA and 2AOB) and the NMR structure of GRB2 in complex
with 3 (PDB ID, 1X0N), key H-bonding interactions with Lys109,
Leu120, and His107 described above and van der Waals contacts with
Phe108, Leu 111, His107, Lys109, and Ser96 were maintained in the
crystal structure of GRB2 with 7. The H- and electrostatic bonding of
GRB2 Arg86 and Ser96 with 3 and 6 seems stronger than that with 7,
and the H- and electrostatic bonding of GRB2 Arg 67 with 3 and 6 is
3

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Bioorganic & Medicinal Chemistry Letters 51 (2021) 128354
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Declaration of Competing Interest
24 Ogura K, Shiga T, Yokochi M, Yuzawa S, Burke Jr TR, Inagaki F. Solution structure of
the Grb2 SH2 domain complexed with a high-affinity inhibitor. J Biomol NMR. 2008;
42:197–207.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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of Grb2-SH2 domain-swapping. Arch Biochem Biophys. 2007;462:47–53.
26 Benfield AP, Teresk MG, Plake HR, DeLorbe JE, Millspaugh LE, Martin SF. Ligand
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Acknowledgements
27 DeLorbe JE, Clements JH, Teresk MG, et al. Thermodynamic and structural effects of
conformational constraints in protein-ligand interactions. Entropic paradoxy
associated with ligand preorganization. J Am Chem Soc. 2009;131:16758–16770.
28 Delorbe JE, Clements JH, Whiddon BB, Martin SF. Thermodynamic and structural
effects of macrocyclization as a constraining method in protein-ligand interactions.
ACS Med Chem Lett. 2010;1:448–452.
This work was supported by 2017 and 2018 Moffitt Cancer Center
Molecular Medicine (MM) Program Innovation Funds, and 2019 Moffitt
Cancer Center Lung Cancer Center for Excellence Debartolo Thoracic
Research Funds. We thank Dr. Zhen Wang for the initial synthesis of
compound 8, and Dylan J. Smith for collecting some NMR and mass
spectrometry data. We also thank the Moffitt Chemical Biology Core for
use of the NMR, mass spectrometry, and protein crystallography facil-
ities supported by National Cancer Institute grant P30-CA76292.
29 Myslinski JM, DeLorbe JE, Clements JH, Martin SF. Protein-ligand interactions:
thermodynamic effects associated with increasing nonpolar surface area. J Am Chem
Soc. 2011;133:18518–18521.
30 Myslinski JM, Clements JH, Martin SF. Protein-ligand interactions: probing the
energetics of a putative cation-π interaction. Bioorg Med Chem Lett. 2014;24:
3164–3167.
31 Yao Z-J, King CR, Cao T, et al. Potent inhibition of Grb2 SH2 domain binding by non-
phosphate-containing ligands. J Med Chem. 1999;42:25–35.
Appendix A. Supplementary data
32 Shi ZD, Lee K, Liu H, et al. A novel macrocyclic tetrapeptide mimetic that exhibits
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2003;310:378–383.
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