Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain

Article abstract of DOI:10.1016/j.ejmech.2013.04.038

Among the EGFRs, HER2 is a major heterodimer partner and also has important implications in the formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be modulated by small molecule ligands and, hence, these protein-protein interactions play a key role in biochemical reactions related to control of cell growth. A peptidomimetic (compound 5-1) that binds to HER2 protein extracellular domain and inhibits protein-protein interactions of EGFRs was conjugated with BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s-indacene). Conjugation of BODIPY to the peptidomimetic was investigated by different approaches. The conjugate was characterized for its ability to bind to HER2 overexpressing SKBR-3 and BT-474 cells. Furthermore, cellular uptake of conjugate of BODIPY was studied in the presence of membrane tracker and Lyso tracker using confocal microscopy. Our results suggested that fluorescently labeled compound 5-7 binds to the extracellular domain and stays in the membrane for nearly 24 h. After 24 h there is an indication of internalization of the conjugate. Inhibition of protein-protein interaction and downstream signaling effect of compound 5-1 was also studied by proximity ligation assay and Western blot analysis. Results suggested that compound 5-1 inhibit protein-protein interactions of HER2-HER3 and phosphorylation of HER2 in a time-dependent manner.

Full text of DOI:10.1016/j.ejmech.2013.04.038

Accepted Manuscript
Design, synthesis and characterization of peptidomimetic conjugate of BODIPY
targeting HER2 protein extracellular domain
Sashikanth Banappagari, Alecia McCall, Krystal Fontenot, M.Graca H. Vicente, Amit
Gujar, Seetharama Satyanarayanajois
PII:
S0223-5234(13)00270-5
10.1016/j.ejmech.2013.04.038
EJMECH 6148
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2013
Revised Date: 15 April 2013
Accepted Date: 18 April 2013
Please cite this article as: S. Banappagari, A. McCall, K. Fontenot, M.G.H. Vicente, A. Gujar, S.
Satyanarayanajois, Design, synthesis and characterization of peptidomimetic conjugate of BODIPY
targeting HER2 protein extracellular domain, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.04.038.
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ACCEPTED MANUSCRIPT
Highlights
*A peptidomimetic targeting HER2 protein was conjugated with fluorescent label BODIPY.
*Cellular uptake of the conjugate was studied by confocal microscopy
*Inhibition of protein-protein interaction was studied by proximity ligation assay
*Designed compound inhibited HER2-HER3 interaction and inhibited phosphorylation
*Fluorescent conjugate designed is useful to study EGFR trafficking

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Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting
HER2 protein extracellular domain
Sashikanth Banappagari,1 Alecia McCall2, Krystal Fontenot,2 M. Graca H. Vicente,2 Amit
Gujar,1 Seetharama Satyanarayanajois.1*.
1Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe,
Monroe LA 71201
2Department of Chemistry, Louisiana State University, Baton Rouge LA, 70803, USA
*Corresponding author
Seetharama D. Satyanarayanajois
Associate Professor
Department of Basic Pharmaceutical Sciences
University of Louisiana at Monroe
1800 Bienville Drive
Monroe, LA 71201
Tel: (318)342-1993
Fax:(318)342-1737
e-mail: jois@ulm.edu
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ABSTRACT
Among the EGFRs, HER2 is a major heterodimer partner and also has important implications in
the formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be
modulated by small molecule ligands and, hence, these protein-protein interactions play a key
role in biochemical reactions related to control of cell growth. A peptidomimetic (compound 5-1)
that binds to HER2 protein extracellular domain and inhibits protein-protein interactions of
EGFRs was conjugated with BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-
indacene). Conjugation of BODIPY to the peptidomimetic was investigated by different
approaches. The conjugate was characterized for its ability to bind to HER2 overexpressing
SKBR-3 and BT-474 cells. Furthermore, cellular uptake of conjugate of BODIPY was studied in
the presence of membrane tracker and Lyso tracker using confocal microscopy. Our results
suggested that fluorescently labeled compound 5-7 binds to the extracellular domain and stays
in the membrane for nearly 24 h. After 24 h there is an indication of internalization of the
conjugate. Inhibition of protein-protein interaction and downstream signaling effect of
compound 5-1 was also studied by proximity ligation assay and western blot analysis. Results
suggested that compound 5-1 inhibits protein-protein interactions of HER2-HER3 and
phosphorylation of HER2 in a time-dependent manner.
Key words: BODIPY, Breast cancer, EGFR, Protein-Protein interactions, HER2,
peptidomimetic, Phosphorylation
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1.
Introduction
Human epidermal growth factor receptors (EGFRs) are important molecules in signal
transduction processes in cells and provide the signal for cell growth, differentiation, and
motility. There are four well known EGFRs, namely, EGFR/HER1/ERBB1, HER2/ERBB2,
HER3/ERBB3, and HER4/ERBB4. HER1 and HER2 have been associated with various cancers
through their overexpression on the cell surfaces of tumors[1-3]. Each receptor exists as a
monomer, consisting of an extracellular domain (containing four domains (I-IV)), a
transmembrane domain, a cytosolic tyrosine-kinase-containing domain, and the regulatory
domain, through which cell signaling to the nucleus occurs [3-7]. EGFRs interact with one
another upon ligand binding to their extracellular domains, and form homo- and heterodimers [3,
8, 9]. Formation of these heterodimers is important in generating signals for cell growth and
differentiation. Other possible dimers such as EGFR-HER2, HER2-HER3, and HER2-HER4
have been proposed in the literature [10]. Blockade of protein-protein interactions (PPI) of the
extracellular domains of EGFRs ultimately leads to control of cell growth [11-14]. Among the
EGFRs, HER2 is a major heterodimer partner and also has important implications in the
formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be
modulated by small molecule ligands and, hence, these protein-protein interactions play a key
role in biochemical reactions related to control of cell growth [13, 15-18]. Antibodies and
affibody molecules targeting HER2 protein are investigated as probes or as imaging agents with
radioactive probes, quantum dots, and fluorescent probes [19, 20]. Peptides designed to target the
extracellular domain (ECD) of HER2 have been reported to interfere with the dimer interface of
HER2 with EGFR [21-23]. Other small molecules have been reported to interfere with the EGFR
extracellular domain [24]. These molecules were designed for therapeutic or imaging purposes.
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Some of these molecules bind to HER2 protein with high affinity, but do not interfere with PPI
[23] . Our approach is to use peptidomimetics and their conjugates to study hot spots in PPI in
the ECD of EGFRs. Among the ECD of EGFRs, domain II is well characterized. Domain IV is
close to the membrane environment and dynamic in nature; it is not well characterized because
in the crystal structure the electron density of domain IV is not well defined [25, 26] . Domains
IV of HER2 and EGFR are known to interact with each other. Thus, we chose to study domain
IV PPI interaction. In our preliminary study, we have shown that a peptidomimetic, namely,
compound 5-1 (Scheme 1), modulates dimerization of EGFR-HER2/HER2-HER3 [27-30]. This
work focuses on conjugation of the fluorescent probe 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-
diaza-s-indacene (BODIPY) to compound 5-1 to modulate PPI interactions of EGFRs [31-34].
Furthermore, we characterized the binding and cellular uptake of BODIPY conjugate of 5-1
(compound 5-7) in HER2-overexpressing breast cancer cell lines. Inhibition of protein-protein
interaction of the compound 5-1 was also studied by proximity ligation assay (PLA). Our results
suggested that fluorescently labeled compound 5-7 binds to the extracellular domain and stays
in the membrane for nearly 24 h. After 24 h there is an indication of internalization of the
conjugate.
2. Results
2.1 Synthesis of BODIPY-HERP5 Conjugates
The synthesis of BODIPY-compound 5-1 conjugate was performed using two methods:
1) solution-phase conjugation of peptomimetics 5-1 and 5-2 to 5-3 (Scheme 1) and 2) solid-phase
conjugation using 5-6 (Scheme 2).
The direct conjugation of peptidomimetic 5-1 to commercially available compound 5-3
under basic conditions (Et3N) gave low yields of conjugate due to the competing polymerization
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reaction of peptomimetic 5-1. Therefore, a tert-butyl protecting group was introduced at the C-
terminus of 5-1, which is stable under the basic conditions needed for conjugation. The tert-
butyl ester can be formed by several methods, including strong acidic conditions.
Mechanistically, the reaction is acid catalyzed with sulfuric acid, then tertiary butanol is added to
complete the esterification. To prevent water from forming and causing hydrolysis of the ester,
anhydrous magnesium sulfate was added. Compound 5-2 was obtained in 54% yield. Solution-
phase conjugation of 5-2 to compound 5-3 in DMF for 72 h gave 20% of tert-butyl protected
compound 5-4. Due to the low overall yield of this method, solid-phase conjugation was then
investigated.
For the solid-phase conjugations, compound 5-1 was prepared by SPPS on two
different resins, the Rink amide resin and the Wang resin. The Rink amide resin provided the
final conjugate with a C-terminal amide functionality whereas the Wang resin gave
product 5-7 with a carboxyl group at the C-terminal (similar to active compound 5-1) that
was used in the biological studies. Conjugation of BODIPY 5-3 to compound 5-6 in Et3N and
DMF proceeded for a total of 72 h. The progress of the reaction was monitored by mass
spectrometry. Once the mass of the conjugation was confirmed, the entire conjugate was cleaved
from the resin using cleavage cocktail, dried, and washed with cold ether to remove scavangers.
Conjugation of 5-3 to compound 5-6 in Et3N and DMF gave the target conjugate 5-7 in 45%
yield after purification by preparative TLC (10% MeOH/90% DCM) (Fig. 1)
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2.2 PLA assay to study protein-protein interaction and its inhibition by compound 5-1
Any protein-protein interaction can be detected by PLA probes if the two proteins are in
proximity of less than 16 nm [35]. One of our objectives is to design compounds to inhibit
protein-protein interactions of EGFRs, in particular, EGFR-HER2 and HER2-HER3. PLA assay
is used to study protein-protein interactions in native cellular environment [35, 36], and thus can
be used to study HER2-HER3 interaction and its disruption by compound 5-1. This method
provides direct evidence of modulation of HER2-HER3 interaction by peptides we have
designed. PLA is an antibody-based method in which two proteins (or antigens) are
immunolabeled first with two primary antibodies and then with different species-specific
secondary antibodies conjugated to complementary oligonucleotides [36]. When two antibody
molecules are in close proximity (<16 nm), the complementary DNA strands can be ligated,
amplified, and visualized with a fluorescent probe as distinct fluorescent spots. Each fluorescent
spot corresponds to a heterodimer on a breast cancer cell. Using PLA, we could observe HER2-
HER3 heterodimers in SKBR3 cells by the probes, seen as red spots. If compound 5-1 inhibits
HER2 heterodimerization, there should be no red spots or relatively fewer spots compared to the
control. SKBR-3 cells were treated with compounds 5-1 and a control peptide for 24 h. Confocal
images of the cells without any compound 5-1 treatment clearly show many HER2-HER3
heterodimers on SKBR-3 cells (Fig. 2A). Treatment with a control peptide did not inhibit HER2-
HER3 heterodimers (Fig. 2B). A sub-optimum dose of compound 5-1 did not inhibit HER2-
HER3 heterodimerization to a significant extent (Fig. 2C) whereas optimum doses of compound
5-1 at 0.8 µM inhibited HER2-HER3 heterodimerization significantly (Fig. 2D). This clearly
suggests that compound 5-1 designed is a potent inhibitor of HER2 heterodimerization with
HER3.
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2.3 Time-dependent inhibition of phosphorylation of HER2 kinase
HER2 heterodimerization causes transphosphorylation and activation of the downstream
signaling cascade, resulting in cell growth and proliferation [3, 5, 37]. Thus, inhibiting
phosphorylation of HER2-mediated dimers plays a major role in inhibiting cell growth and
proliferation. To evaluate the effect of compound 5-1 on the dimerization of full-length EGFRs
in the native form in HER2-overexpressing breast cancer cell lines, the effect of compound 5-1
on the phosphorylation of HER2 kinase domain in BT-474, SKBR-3 and MCF-7 cells was
investigated using Western blot. Inhibition of the receptor heterodimerization or association in
cells by compound 5-1 should decrease the phosphorylation level of HER2 protein.
Phosphorylation was detected using p-HER2, an antibody that specifically binds to the
phosphorylated HER2 protein. Fig. 3 represents the Western blot analysis of the phosphorylation
level in treated and untreated BT-474, SKBR3, and MCF-7 cells, respectively. Cells without
compound 5-1 and AG-825 (0.38 µM, a kinase inhibitor, not shown) [37, 38] were used as a
control. Comparison of the inhibition of phosphorylation and control suggested that compound
5-1 showed significant inhibition of phosphorylation of HER2 in BT-474 and SKBR-3 cells in a
time-dependent manner. A significant inhibition of HER2 phosphorylation was observed after 18
h of treatment with compound 5-1. These results clearly suggest that binding of compound 5-1 to
ECD affects the intracellular transphosphorylation and, hence, the signaling process. MCF-7
cells that do not show overexpression of HER2 protein were used as a control, and no
phosphorylation of HER2 was detected in those cells.
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2.4 Cellular binding and uptake studies
Compound 5-1 has been shown to bind to the HER2 extracellular domain on BT-474 and SKBR-
3 cells that overexpress HER2 protein. Once compound 5-1 binds, inhibition of EGFR-HER2
and HER2-HER3 dimerization takes place and, hence, disrupts the phosphorylation. However,
the fate of compound 5-1 after the inhibition of PPI of EGFRs is not known. In our earlier report
we have shown that compound 5-1 with free carboxy terminal exhibits antiproliferative activity
against HER2 overexpressing breast cancer cells [27]. Thus, we investigated the cellular binding
and uptake of compound 5-1 by conjugating it with BODIPY (compound 5-7). Time-dependent
uptake was monitored by fluorescence emitted from the SKBR-3 cells at two different
concentrations of compound 5-7 (1 and 10 µM). At 1 µM concentration of the conjugate, there
was a steady increase in fluorescence with respect to time up to 72 h. At 10 µM concentration of
the conjugate, saturation was observed after 48 h. (Fig. 4). These data correlate with the time-
dependent inhibition of HER2 phosphorylation by compound 5-1 (Fig. 3). To further investigate
the importance of the increase in fluorescence and localization of the compound 5-7, cellular
uptake was monitored by confocal microscopy using membrane and Lyso trackers. Confocal
images suggested that, even after 24 h, the majority of compound 5-7 is on the surface or
membrane of SKBR-3 cells (Fig. 5). Images of cells after 48 h of compound 5-7 incubation
using Lyso tracker marker show clearly that part of the conjugate is taken up into the cell as
indicated in Fig. 6. These data suggest that the compound 5-7 binds to the extracellular domain
of HER2 protein on the surface of SKBR-3 cells and possibly is internalized after 48 h. The
exact mechanism of internalization is not clear at this stage.
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3. Discussion
HER2, also known as ERBB2, is found to overexpress in many epithelial malignancies. It is well
established that HER2-mediated heterodimerization plays key roles in the progression of such
cancers [1-10, 17] . It has been reported that EGFR-HER2 and HER2-HER3 heterodimers are
major contributors to mediating the pathological effects of this overexpression in HER2-
overexpressing breast cancer [9, 10, 17]. Inhibition of HER2 heterodimerization contributes
significantly to the treatment of HER2-positive breast cancers. Compound 5-1 that we have
designed has been shown to bind to the HER2 extracellular domain and inhibit protein-protein
interactions. Conjugation of compound 5-1 to BODIPY was achieved using different synthetic
strategies. Although conjugation of fluorescent molecules seems to be quite simple using amine
coupling methods, conjugation to purified compound 5-1 posed a problem. Hence, compound 5-
1 had to be conjugated to the peptide on the resin. The conjugate (compound 5-7) was
characterized by mass spectrometry and UV-fluorescence spectroscopy. Cellular uptake studies
suggested that the compound 5-7 is taken up by HER2-overexpressing cell lines in 48 h and, at a
concentration of 10 µM, saturation in the uptake was observed. At present, the exact mechanism
cannot be proposed for this observed result; multiple mechanisms could be possible for the time-
dependent uptake of the compound 5-7. As the binding site of compound 5-1 is HER2-domain
IV, which is close to the cell membrane, its conjugate, compound 5-7 might be trapped in the
cell membrane after dissociation. Finally, after exerting its action, compound 5-7 might
internalize into the organelles of the breast cancer cell. To determine the possibility of
internalization, cells were incubated with the compound 5-7 for 24 and 48 h and co-localization
of compound 5-7 with membrane and Lyso trackers was observed using confocal microscopy.
The results clearly suggested that the conjugate stays on the cellular surface up to 24 h,
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indicating binding to ECD of HER 2 protein. However, after 48 h, there was an indication of
internalization of the compound 5-7. EGFR receptors are known to undergo internalization after
forming ligand-bound dimers. These dimers are known to be sorted in endosomes and recycled
back to the surface and/or undergo endosomal degradation [39-41]. Our results suggest that
compound 5-7 bound to HER2 protein is retained extracellularly for up to 24 h and, hence, may
not undergo recycling with the receptor. Previous studies have reported that overexpression of
HER2 and EGFR-HER2 heterodimerization completely abolishes the EGFRs degradation
process [42]. However, recent literature suggested that HER2 overexpression and
heterodimerization slows down EGFR internalization and degradation [39-41]. Results from our
studies suggested that compound 5-7 that binds to the HER2 extracellular domain is retained
extracellularly for 24 h, which correlates with a slowdown of the internalization process of
EGFR heterodimers. To show that compound 5-1 inhibits protein-protein interaction, we carried
out PLA assay. Those results clearly suggest that inhibition of dimerization of extracellular
domains of HER2 and HER3 results in inhibition of PPI of HER2-HER3. Full-length EGFRs
have several regions of contact for PPI when they form homo- and heterodimers [3, 5, 9].
Domain II, the dimerization arm, domain IV, the transmembrane domain, and the kinase domain
all form contacts for PPI of EGFRs. Among these, domain II interactions are well understood in
EGFR homodimers, and the antibody pertuzumab, which binds to domain II of HER2 protein,
inhibits PPI of EGFRs [14]. The interaction of domain IV is not well understood. The literature
suggests that domain IV may be involved in stabilizing the PPI of EGFRs. However, the
contribution of domain IV to PPI is very weak. Studies also suggest that domain IV helps to
align the transmembrane domains [43]. The crystal structures of homodimers of EGFR suggest
that hydrophobic residues such as Leu582, Trp584, and Tyr602 participate in PPI of domain IV
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to stabilize the interaction of homodimers of EGFR near the transmembrane [26]. The results
from our current and previous studies [28, 30] clearly suggest that a peptidomimetic that we
designed targets domain IV and inhibits PPI in vitro as seen in the PLA assay. This inhibition in
turn has the pharmacological action of inhibition of phosphorylation of the kinase domain and,
hence, modulation of the signal for cell growth. Furthermore, inhibition of the extracellular
domain of HER2-HER3 inhibits the phosphorylation of HER2 protein in a time-dependent
manner. Western blot analysis of HER2 phosphorylation showed maximum inhibition of HER2
phosphorylation after 24 h of treatment. We have also shown that conjugation of a
peptidomimetic that binds to domain IV of HER2 with a fluorescent label such as BODIPY helps
us to study the molecular mechanism of the signal transduction process. These studies taken
together suggest that a compound 5-7 could be used as a tool to study the binding and
internalization of EGFR receptors and their heterodimerization. The data presented here provide
a new tool to study the EGFRs PPI inhibition and EGFR trafficking.
4. Conclusions
A peptidomimetic (compound 5-1) that binds to HER2 protein extracellular domain and inhibits
protein-protein interactions of EGFRs was conjugated with the fluorescent dye BODIPY.
Synthesis was achieved with conjugation on solid-phase synthesis. PPI inhibition activity of the
compound was evaluated by proximity ligation assay. The PLA assay suggested that the
compound inhibits HER2-HER3 heterodimerization in lower micromolar concentrations
effectively. The downstream signaling effect of PPI inhibition was evaluated by time-dependent
phosphorylation by compound 5-1. Compound 5-1 inhibited phosphorylation significantly within
18 to 24 h. To evaluate the effect of compound on the PPI of EGFR and the fate of the
compound after PPI inhibition, cellular uptake of the newly synthesized BODIPY conjugate of
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compound 5-1 (compound 5-7) was studied by fluorescence plate reader assay and confocal
microscopy with organelle tracers. Compound 5-7 seems to reside in the extracellular region or
in the membrane for up to 24 h; at 48 h, there was an indication of internalization. The
internalization was viewed in terms of EGFR trafficking. However, more detailed studies of the
kinetics of EGFR trafficking are necessary to understand the receptor internalization and
recycling to the surface of cells using fluorescent conjugates. Thus, the conjugates designed here
will be useful tools for studying EGFR trafficking and the effect of inhibition of HER2
heterodimerization on EGFR trafficking and ligand receptor interactions.
5. EXPERIMENTAL PROCEDURES
5.1 General information
All chemicals, biochemicals and solvents were purchased from commercial sources. All peptide
synthesis reagents were purchased from Fisher Scientific or Sigma Aldrich as ACS grade or
peptide synthesis grade solvents. Amino acids were purchased from AnaSpec (Fremont, CA) or
Applied Biosystems (Carlsbad, CA). Beta-amino acid, Fmoc-3-amino-3-(1-naphthyl)-propionic
acid]-OH, was purchased from Chem-Impex International (Wood Dale, IL). Analytical thin-layer
chromatography (TLC) was carried out using polyester backed TLC plates 254 (precoated, 200
ꢀm) from Sorbent Technologies. NMR spectra were recorded on an AV-400 LIQUID Bruker
spectrometer (400 MHz for 1H). 2D TOCSY spectra of compounds 5-1 and 5-7 were
recorded in 500 and 700 MHz Varian NMR spectrometers respectively. The chemical shifts
are reported in δ ppm using the following deuterated solvents as internal references: acetone-d6
2.05 ppm (1H), DMF-d7 8.03 ppm (1H), H2O 90%/D2O 10%. HPLC analyses were carried
out on a Dionex system equipped with a P680 pump and UVD340U detector. MALDI-TOF mass
spectra were recorded on a Bruker ProFlex III mass spectrometer using dithranol as the matrix or
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Bruker UltrafleXtreme (MALDI-TOF/TOF) using 4-chloro-α-cyanocinnamic acid as the matrix;
ESI mass spectra were obtained on an Agilent Technologies 6210 time-of-flight LC/MS with a
quaternary gradient module pump, 2489 UV−visible detector, and fraction collector III.
Analytical HPLC was carried out using a XBridge C18 300 Å, 5 ꢀm, 4.6 mm × 250 mm column
(Waters, USA) and a stepwise gradient. Semipreparative HPLC was carried out using a XBridge
C18 300 Å, 5 ꢀm, 10 mm × 250 mm column (Waters, USA) and a stepwise gradient. The solvent
system for peptides consisted of Millipore water and HPLC-grade acetonitrile/methanol.
Antibodies for Western blot analysis were obtained from Abcam, Inc. (Cambridge, MA).
Antibodies and wash buffers A and B for PLA were obtained from Axxora, LLC (Farmingdale,
NY) and nanoTools (San Diego, CA). Control peptide was custom synthesized by Polypeptide
laboratories (San Diego, CA).
5.2 Chemistry
5.2.1 Synthesis of compound 5-1
The peptidomimetic (compound 5-1) was synthesized by different methods to determine the
efficiency of conjugation. Conjugation of BODIPY to compound 5-1 was attempted using different
methods: 1) conjugation to derivative compound 5-2 in solution phase (Scheme 1), 2) solid-phase
conjugation using on Wang resin and on Rink amide resin. Since biological activity of compound 5-
7 sythesized using Wang resin was evaluated. Details of synthesis of Rink amide resin based
conjugate is provided elsewhere (Ph.D dissertation, Alecia McCall, Louisiana State University April
2012).
Peptidomimetic compound 5-1 was synthesized by manual solid-phase synthesis using
standard procedures[44]. The peptide was synthesized on Wang resin on a 0.2 mmol scale using
the Fmoc strategy of solid-phase peptide synthesis. A four fold excess of the L-Fmoc-protected
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amino acids was coupled using PyAOP/HOBT or HATU as the coupling and activating agents.
Resins were allowed to swell in DMF for 1 h, and the Fmoc-protecting group was removed from
the resin. For coupling Arg to the growing peptide chain, double coupling was performed. The
Fmoc-protecting group was cleaved with 93% DMF, 5% piperidine, and 2% DBU once for 3
min, once for 5 min, and once for 10 min. This was followed by washing the peptide several
times with DMF after which the next amino acid was coupled. The Fmoc group from
phenylalanine was not cleaved until the peptide was ready for use. The peptide was then washed
with DMF (3 × 30 s) and DCM (3 × 30 s), respectively, and dried under high vacuum for 24 h.
The peptide was then cleaved using a cleavage cocktail consisting of TFA/liquefied
phenol/water/Tris (88:5:5:2) for 5 h. This cocktail was then precipitated into anhydrous ethyl
ether (–80 °C) and centrifuged. The precipite was washed three times with anhydrous ethyl ether
(–80 °C) and then dissolved in water and acetonitrile and lyophilized. Reversed-phase HPLC
analysis, mass spectrometry, and NMR studies were performed to evaluate the purity of
compound 5-1. High resolution mass spectrum and 1H 2D NMR of compound 5-1 with
assignments are provided as supporting information.
5.2.2 Synthesis of tert-butyl Peptomimetic (5-2): In a small round bottom flask, anhydrous
magnesium sulfate (0.232 mg, 0.00193 mmol) in 3 mL of dry DCM was stirred vigorously for 10
min. Excess concentrated sulfuric acid (92.00 mg, 0.9380 mmol) was added to the flask. The
mixture was stirred for 1 h. Compound 5-1 (0.25 mg, 4.82×10-4 mmol) was then added to the
mixture (Scheme 1). The flask was sonicated to ensure solubility and stirred for an additional 5
min before adding tertiary butanol (0.228 mL, 0.00241 mmol). The flask was stoppered and
stirred for 24 h at 25oC under N2 gas. The reaction was quenched with saturated sodium
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bicarbonate (3 mL) by adding this to the reaction flask until all of the magnesium sulfate had
dissolved. The organic phase was separated, washed with brine, dried with Na2SO4, and purified
1
by preparative TLC to afford the t-butyl protected compound (5-2) in 54% yield (0.15 mg). H
NMR (400 MHz, acetone-d6) δ ppm 1.28 (br, 9H), 1.51-1.62 (m, 2H), 1.79 (m, 2H), 3.30 (m,
2H), 4.50-4.63 (m, 1H), 5.34 ( 1H), 6.24-6.31 (m, 1H), 7.00-7.10 (m 5H), 7.15 (1H), 7.32 (1H)
7.31 (d, J=4.26 Hz 1H), 7.42-7.80 (m 7H), 7.93 (d, J=7.31 Hz 1H), 8.15 (br, 1H), 8.22 (m,
1H). Arg side chain guanidine group protons (4H) were broad, and phenyl alanine beta
protons (2H) were under the solvent peak around 3 ppm, and hence could not be identified.
MS (MALDI-TOF): m/z 597.428. Calcd. for C32H42N6O4: 597.316 [M+Na].
5.2.3 Synthesis of BODIPY succinimidyl ester tert-butyl Arg-[3-amino-3-(1-napthyl)-propionic
acid]-Phe (5-4) (Scheme 1): Compound 5-3 was purchased from Life Technologies (Carlsbad,
CA). To a small round bottom flask was added 5-3 (0.826 mg, 0.00198 mmol) in peptide
synthesis grade DMF (1 mL). Compound 5-3 was sonicated to ensure solubility. Six (6)
equivalents of triethylamine (1.65 µL, 0.011 mmol) were added to the flask. Compound 5-3 was
activated for 4.5 h under inert conditions (N2 gas) before compound 5-2 (2.3 mg, 0.00198 mmol)
in 0.5 mL peptide synthesis grade DMF was added to the flask. Conjugation took place for 72 h
under nitrogen. Confirmation of mass was observed by MS (ESI-MS): m/z 820.737. Calcd. for
C44H51BF2N8O5 820.734 [M-tBu].
5.2.4 Conjugation of BODIPY 5-3 to Arg-[3-amino-3-(1-napthyl)-propionic acid]-Phe on Wang
Resin (5-7): Synthesis of compound 5-7 (Scheme 2) was achieved by routine solid-phase
peptide synthesis. To a reaction vessel compound 5-3 (0.0021 g, 0.0050 mmol) was added and
solubilized in 0.9 mL DMF. Triethylamine (0.006 mL, 0.043 mmol) was added to the vessel and
stirred to activate the ester (Scheme 2). The reaction continued for 1 h. This solution was added
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to the peptide synthesis chamber containing 5-6 (0.0050 g, 0.00479 mmol) (Scheme 2). The
reaction proceeded with shaking for approximately 72 h under N2. The beads were then washed
with DMF 4 times until the filtrate was colorless to remove residual compound 5-3. A cleavage
cocktail consisting of 1 mL of TFA (88%), liquefied phenol (5%), triisopropylsilane (2%), and
water (5%) was added to the resin beads for 4 h. The resin was filtered and washed with TFA (3
x 1 mL). Filtrates were combined, evaporated, and washed with cold anhydrous ether.
Purification was conducted by preparative TLC (10% MeOH/90% DCM) and analytical HPLC
1
(ACN/H2O) to yield 45%-65% of conjugate. H NMR (700 MHz, DMF-d7) δ ppm 8.75 (br,
1H), 8.14 (d, J=7.0Hz 1H), 7.99 (d, J= 7.52 1H), 7.88 (br, 1H), 7.60-7.80 (m, 4H), 7.31(br,
1H), 7.24(m, 3H), 6.80-7.00(m,6H), 6.41-6.62(br. 2H), 6.30(br, 1H), 6.00 (br, 1H), 4.45(m,
1H), 4.31 (m, 1H), 3.21-3.42(br, 6H), 2.80-2.84 (m, 3H), 2.42(m, 3H), 1.90-2.00 (m, 2H), 1.61-
1.77 (m, 3H), 1.45-1.50 (m, 3H), 1.21-1.60 (m, 6H). C-terminal carboxylic acid proton were
broad and were not observed. MS (ESI-TOF): m/z 821.408. Cald. for C44H51BF2N8O5
821.411 [M+H] (Fig. 1). UV-vis (DMSO) λmax (nm) (ε, L mol−1 cm−1): 485 nm (4.49E+05), 351
(5.90E+05).
5.3 Proximity ligation assay
Proximity ligation assays (PLA) were performed on breast cancer cells SKBR-3 that overexpress
HER2 protein [35, 36]. PLA assay on breast cancer cells was performed by coating 5 × 104 cells
onto each well in an 8-chambered plate. The plate was incubated for 24 h at 37 oC. Compound 5-
1 was added at two different concentrations (0.5 and 0.8 µM). A control peptide (supporting
information) was also added at 1 µM concentration. After 24 h, the medium was removed and
the wells were washed 3 times with 500 ꢀL of PBS. The cells were fixed by adding 500 ꢀL of
o
ice-cold methanol for 5-8 min and incubating the plate at –20 C. The methanol was discarded
16

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and immediately 500 ꢀL of PBS was added twice to the wells to prevent cell dehydration. The
cells were then blocked with 200 ꢀL of 5% BSA for 1 h in a humidity chamber. The blocking
solution in the wells was discarded, and 200 ꢀL of 1:400 dilutions of primary antibodies (both
HER2 (anti-rabbit) and HER3 (anti-mouse)) in 2% BSA were added to the wells. The 8-
o
chambered well plate was then incubated at 4 C in a humidity chamber overnight. Primary
antibodies were discarded and the plate was washed twice with wash buffer (from PLA kit) for 5
min. To the cells, two PLA probes (anti-mouse and anti-rabbit) at 1:5 dilution in 2% BSA were
added and incubated for 1 h at 37 oC in a humidity chamber. Slides were washed with wash
buffer for 5 min twice, and ligation stock at 1:5 dilution in high purity water was added and
incubated for 30 min at 37 oC. The slides were washed again with wash buffer, amplification
solution was added, and the slide was incubated for 2 h at 37 oC. Slides were then washed twice
with wash buffer for 10 min. The slides were dried at room temperature, mounted with mounting
medium (Duolink II), and stored at –20 oC until confocal images were taken. Cells were viewed
with a confocal microscope (LSM 5 PASCAL). Images were taken at 40X as wells and at 60X
under oil immersion.
5.4 Time-dependent inhibition of phosphorylation of HER2 kinase
BT-474 cells were maintained in RPMI-1640 supplemented with pen-strep 1 mL/100 mL, insulin
1 mg/100 mL, and 10% FBS. SKBR-3 cells were maintained in McCoy’s medium supplemented
with penstrep 1 mL/100 mL, insulin 1 mg/100 mL, and 10% FBS. After confluency, cells were
trypsinized and plated in T-75 flasks in triplicate. After 24 h, the cells were either treated with
compound 5-1 (0.4 µM) or left untreated. Cells with compounds were incubated at 37 ºC and 5%
CO2. After the incubation period, cells were trypsinized, collected in 15 mL tubes, and
centrifuged at 200 ×g. To the pellet, 200 µL of cell lysis buffer containing 20 µL of protease
17

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inhibitor and 20 µL of phosphatase inhibitor was added. Tubes were vortexed for 1 min and then
kept on ice for 10 min. This process was repeated twice. Cells were subjected to centrifugation at
high speed (450 ×g) and the supernatant was collected in separate tubes and stored at –80 ºC
until further use. Protein concentration in the tubes was analyzed using Bradford’s assay. 40 ꢀg
of protein from each sample was loaded on 10% SDS-polyacrylamide minigels. The gels were
run at 30 mA, 125 V and then transferred at 30 V for 12–16 h at 4 ºC onto a nitrocellulose
membrane. After transfer, the membranes were blocked with 5% milk for 1 h. The nitrocellulose
membranes were then probed with specific primary antibodies against p-HER2 diluted 1:3000 in
5% milk for 15 h at 4 °C. After incubation with primary antibody, the membranes were washed
five times with PBS and then incubated with secondary antibody to p-HER2 (anti-rabbit
secondary antibody) diluted 1:2000 in 5% milk with 0.001% Tween 20 for 1 h. The membranes
were washed five times with PBS, and proteins were visualized using the Super Signal enhanced
chemiluminescence kit (Pierce, Rockford, IL) [37] . The Kodak Gel Logic-1500 imaging system
(Carestream Molecular Imaging, New Haven, CT) was used to visualize the luminescent
proteins. All experiments were repeated at least three times. A representative Western blot image
was used for the final representation. The visualization of β-actin was used to ensure equal
sample loading in each lane. Total HER2 protein was detected using an antibody to the HER2
extracellular domain (Abcam, Cambridge, MA).
5.5 Binding and cellular uptake of compound 5-7
Peptidomimetic compound 5-1 was conjugated with BODIPY dye to study its interaction with
HER2 protein. SKBR-3 cells were plated at 10,000 per well in a 96-well plate and allowed to
grow for 24 h. Compound 5-7 stock solution was diluted to 1 and 10 µM in McCoy’s medium
and added to the cells. Compound 5-7 was added to the 96-well plates coated with SKBR3, and
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o
the plates were incubated at 37 C. After time points of 0, 12, 24, 36, 48 and 72 h, the medium
with compound 5-7 in the plates was removed, and the cells were washed with PBS twice. The
fluorescence of compound 5-7 was determined by reading its fluorescence emission at 490
nm/530 nm (excitation/emission) using a BioTek plate reader (Cary, NC). Similar experiments
were conducted using chamber slides, and images of cells with fluorescence were obtained using
a confocal microscope. To the 8-well plates coated with SKBR-3, compound 5-7 at a
concentration of 0.4 µM was added, and the plates were incubated at 37 oC. After time points of
24 and 48 h, the medium with compound 5-7 in the plates was removed, and the cells were
washed with PBS twice. The cells were then fixed by adding 500 ꢀL of ice-cold methanol and
incubating the plate at –20 oC for 5 min. The methanol was discarded and immediately 500 ꢀL of
PBS was added twice to the wells to prevent cell dehydration. Membrane and lyso trackers were
diluted in serum-free medium and added to the wells treated with compound 5-7 as well as to
the wells without any treatment. The plates were incubated for 5 min (membrane tracker) and 30
min (Lyso tracker) at 37 oC. After the incubation period, the wells were washed with PBS twice,
dried, and mounted with mounting medium for imaging [45]. Cells were viewed with a confocal
microscope (LSM 5 PASCAL). Images were taken at 40X under oil immersion with argon and
HeNe lasers. Images were analyzed with ZEN 2009 software.
Acknowledgments
Research reported in this publication was supported by an Institutional Development Award
(IDeA) from the National Institute of General Medical Sciences of the National Institutes of
Health under grant number P20GM103424 and Louisiana Board of Regents (NSF(2010)-
PFUND-219) (SJ). The content is solely the responsibility of the authors and does not
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ACCEPTED MANUSCRIPT
necessarily represent the official views of the National Center for Research Resources or the
National Institutes of Health. The authors thank Dr. Karen Briski, Chair, Department of Basic
Pharmaceutical Sciences, College of Pharmacy at ULM for the use of confocal microscopy
facility. Department of Chemistry, Louisiana State University, Baton Rouge, LA, is
acknowledged for mass and NMR spectra of compounds. Authors would like to thank Ms.
Nancy Harmony for help with editing the manuscript.
Supporting Information Available: Schematic diagram of compound 5-1, 5-5, control
peptide, elemental analysis, mass spectra, HPLC of compounds, 1H 2D TOCSY NMR
spectra and fragmentation data from mass spectral analysis of the compounds.
ABBREVIATIONS USED
ACN, acetonitrile; BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; BSA, bovine serum
albumin; ECD, extracellular domain; 6-Cl-HOBt, 6-chloro-1-hydroxy-1H-benzotriazole; DMF,
dimethylformamide; DCM, dichloromethane; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; EGFR,
Epidermal growth factor receptor; Et3N, triethylamine; HATU, O-(7-Azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; HER, human epidermal growth factor
receptor; HRMS (ESI), high-resolution mass spectrometry (electro-spray ionization); LC/MS,
liquid
chromatography-mass
spectrometry;
MALDI-TOF,
matrix-assisted
laser
desorption/ionization-time of flight; MeOH, methanol; PBS, phosphate-buffered saline; p-HER2,
phosphorylated HER2; PLA, proximity ligation assay; PPI, protein-protein interactions; PyAOP,
7-azabenzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate; SDS, sodium
dodecyl sulfate; SPSS, solid-phase peptide synthesis; TFA, trifluoroacetic acid.
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Schemes, Figure captions
Scheme 1: Synthesis of 5-2 and 5-4 (tert-Butyl ester terminated conjugate).
Scheme 2: Synthesis of compound 5-7 using Wang resin.
Fig. 1. High resolution mass spectrum (HR-MS) of compound 5-7.
Fig. 2. Inhibition of HER2-HER3 interaction evaluated by PLA assay (Duolink II). A) SKBR-3
cells without any treatment. Red dots indicate heterodimerization of HER2-HER3 proteins.
Notice the overexpression and dimerization of HER2 with HER3. B) SKBR-3 cells treated with
a control peptide. Notice that HER2-HER3 dimerization is not inhibited. C) and D), 0.5 µM and
0.8 µM compound 5-1 treated SKBR-3 cells. Compound 5-1 inhibited HER2-HER3 dimerization
indicated by the significant reduction in the number of red dots.
Fig. 3. Western blot analysis of phosphorylated HER2. BT-474, SKBR3 cells that overexpress
HER2 protein upon treatment with compound 5-1 (0.4 µM) showed decreased level of
phosphorylated HER2 over time. Total HER2 protein is also shown. A time dependent inhibition
of HER2 phosphorylation by compound 5-1 is observed in BT-474 and SKBR3.
Fig. 4. Time-dependent cellular binding and uptake of compound 5-7 at two concentrations.
Fig. 5. Cellular localization of compound 5-7 monitored by membrane tracker. A) Membrane tracker, B)
compound 5-7, 24 hr, C) overlap 24 hrs. D) membrane tracker 48 hrs, compound 5-7, 48 hrs, F) Overlap.
Fig. 6. Cellular localization of compound 5-7 monitored by Lyso tracker. A) Lyso tracker, B) compound
5-7, 24 hr, C) overlap 24 hrs. D) Lyso tracker 48 hrs, compound 5-7, 48 hrs, F) Overlap.
27