125I-radiolabeled morpholine-containing arginine-glycine- aspartate (RGD) ligand of αvβ3 integrin as a molecular imaging probe for angiogenesis

Article abstract of DOI:10.1021/jm2016232

In this paper, using a hybrid small-animal Micro SPECT/CT imaging system, we report that a new ¹²⁵I-Cilengitide-like RGD-cyclopentapeptide, containing d-morpholine-3-carboxylic acid, interacts in vivo with α_vβ₃ integrin expressed by melanoma cells. Images clearly show that the ¹²⁵I-compound has the capacity to monitor the growth of a melanoma xenograft. Indeed, retention of the labeled ligand in the tumor mass has a good tumor/background ratio, and a significant reduction of its uptake was observed after injection of unlabeled ligand. These results suggest that the use of ¹²⁵I-labeled morpholine-based RGD-cyclopentapeptides targeting α_vβ₃ positive tumors may play a role in future therapeutic strategies.

Full text of DOI:10.1021/jm2016232

Article
pubs.acs.org/jmc
125I-Radiolabeled Morpholine-Containing Arginine−Glycine−
Aspartate (RGD) Ligand of αvβ3 Integrin As a Molecular Imaging
Probe for Angiogenesis
Francesca Bianchini,†,‡ Nicoletta Cini,§ Andrea Trabocchi,∥,# Anna Bottoncetti,§ Silvia Raspanti,#
Eleonora Vanzi,#,# Gloria Menchi,∥,# Antonio Guarna,*,∥,# Alberto Pupi,*,#,#,‡ and Lido Calorini*,†,‡
†Department of Experimental Pathology and Oncology, University of Florence, Viale Morgagni 50, 50134 Florence, Italy
‡Istituto Toscano Tumori (ITT), Via T. Alderotti 26N, 50139 Florence, Italy
§Azienda Ospedaliera Universitaria Careggi (AOUC), Largo Brambilla 3, 50134 Florence, Italy
∥Department of Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia 13, 50019 Sesto Fiorentino, Florence, Italy
#Interdepartmental Center for Preclinical Development of Molecular Imaging (CISPIM), University of Florence, Viale Morgagni 85,
50134 Florence, Italy
#Department of Clinical Physiopathology, Nuclear Medicine Unit, University of Florence, Viale Morgagni 85, 50134 Florence
S
* Supporting Information
ABSTRACT: In this paper, using a hybrid small-animal Micro SPECT/CT imaging system, we report that a new 125I-
Cilengitide-like RGD-cyclopentapeptide, containing D-morpholine-3-carboxylic acid, interacts in vivo with αvβ3 integrin
expressed by melanoma cells. Images clearly show that the 125I-compound has the capacity to monitor the growth of a melanoma
xenograft. Indeed, retention of the labeled ligand in the tumor mass has a good tumor/background ratio, and a significant
reduction of its uptake was observed after injection of unlabeled ligand. These results suggest that the use of 125I-labeled
morpholine-based RGD-cyclopentapeptides targeting αvβ3 positive tumors may play a role in future therapeutic strategies.
angiogenic property, which is under investigation in phase II4
INTRODUCTION
■
and III5 for the treatment of glioblastoma.
As pointed out by the Food and Drug Agency,1 a major
problem with the health system of western societies is the
slowness of drug development. This is because of the increasing
cost of new drug pipelines which at present exceeds 1.5 billion
dollars.2 Use of biomarkers as molecular imaging tools in early
nonclinical and clinical development provides a more informed
scientific basis for the design of pivotal trials and has been
already proven useful in simplifying the development of target
therapies.3 Indeed, the method implemented in preclinical
phases of drug development by molecular imaging can be
translated into clinical trials. In addition, using biomarkers in
molecular imaging of small-animal models has become a new
important scientific field because the animals can be
sequentially used but not sacrificed. At the same time, new
molecular imaging technology is extending the sensitivity for
defining treatment strategies. Among these strategies, new
tumor therapies include Cilengitide, a RGD (arginine−
glycine−aspartic acid) mimetic integrin inhibitor with anti-
Integrins are heterodimeric glycoprotein transmembrane
receptors consisting of various combinations of α and β
subunits. These receptors regulate biological processes such as
apoptosis, cell adhesion and migration, cell differentiation and
angiogenesis, and are key points to determine invasiveness and
metastatic dissemination of tumor cells.6
Integrins on the surface of tumor cells are quite specific and
dictate tumor progression. High expression of integrins
characterizes various malignant tumors, such as breast and
prostate tumors, ovarian carcinoma, glioblastoma, and malig-
nant melanoma. During the transition from a benign
melanocytic lesion to a metastatic malignant melanoma,
melanocytes undergo a series of changes in the expression of
cell-surface integrins. Among these integrins, the vitronectin
Received: November 30, 2011
Published: May 23, 2012
© 2012 American Chemical Society
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Figure 1. Morpholine-based RGD-cyclopeptides: D-Mor-containing compound 1, D-Mor-D-Tyr-containing compound 2, 127I-D-Mor-D-Tyr-
containing compound 3, 125I-D-Mor-D-Tyr-containing compound 125I-3.
receptor αvβ3 plays a fundamental role in melanoma growth
and dissemination. αvβ3 integrin overexpression is associated
with transition of melanoma cells from the radial to vertical
growth phase and characterizes cells at the invasive front of a
tumor.7 In particular, αvβ3 integrin favors the localization of
MMP-2 on the surface of invasive tumor cells,8 and it has been
demonstrated that the expression of membrane type-1 MMP
on the cell surface requires αvβ3 integrin.9 A recent analysis of
the gene-expression profile of 34 vertical growth phase of
human melanoma indicates that proteins of epithelial-
mesenchymal transition (EMT) program, namely αvβ3 integrin
and MMP-2, are significantly correlated with the metastatic
disease.10
of commercially available iodination reagents. Molecules
labeled with iodine represent a convenient tool for animal
models up to humans because substitution of 125I with 123I
(159-KeV γ rays with half-life 13.2 h) provides a probe with a
path-length suitable for imaging in humans. Furthermore,
molecular imaging of iodine-labeled compounds by PET can be
accomplished with the positron emitter 124I, and the translation
to radionuclide therapy using 131I (emitting β- and γ-rays) can
be easily assessed. High-resolution SPECT systems for in vivo
imaging of 125I-labeled biomolecules are important for
developing new imaging probes for diagnostic and therapeutic
approaches.
It is noteworthy that the expression of αvβ3 integrin is also
characteristic of endothelial cells of tumor vasculature and
participates in tumor angiogenesis. Recently, together with αvβ3
integrin, an additional RGD integrin, the fibronectin receptor
α5β1, which plays a role in tumor angiogenesis, has been
found.11
The relevance of RGD-dependent integrins in tumor
progression has stimulated the search of RGD mimetics to
target growth and dissemination of cancer cells and to inhibit
tumor angiogenesis.12
The interaction between natural ligands and the RGD
recognition site of integrins can be inhibited by antagonists that
contain the RGD sequence. Inhibition of different RGD-
integrins can be modulated by the sequence and the structure
of antagonists containing or mimicking the RGD sequence.
Recently, many efforts have been dedicated in developing
PET13 and SPECT14 radiopharmaceuticals addressing the RGD
moiety.
RESULTS
■
We recently synthesized and tested a RGD-based cyclo-
pentapeptide containing D-morpholine-3-carboxylic acid
(Mor) as a replacement for N-methyl-valine as found in
Cilengitide (Figure 1, compound 1). This compound expressed
high binding capacity for αvβ3/αvβ5 integrins.15 In this work, we
have modified compound 1 by replacing a D-Phe with a D-Tyr
(compound 2) and introducing an iodine atom on the aromatic
ring of D-Tyr (compound 3) with the aim of assessing whether
the modified Cilengitide-like inhibitor 3 could be suitable for
SPECT imaging using 125I as the radionuclide. We have also
demonstrated that the morpholine-based RGD-cyclopentapep-
tides 1−3 interact with αvβ3 integrin in vitro and in vivo, and
that the 125I-labeled compound 125I-3 may be used to monitor
melanoma xenografts with a hybrid small-animal Micro
SPECT/CT imaging system.
Synthesis. Compound 2 was prepared as previously
reported15 with direct labeling with NaI (1:1 molar ratio)
using chloramine-T as the oxidizing reagent in phosphate
buffered saline solution (PBS). The reaction was quenched
with sodium metabisulfite, and the crude product was purified
by semipreparative HPLC to give compound 3 (4.7 mg, 32%).
Compound 125I-3 was prepared starting from compound 2 and
Na125I (18:1 molar ratio) (as for compound 3). The crude
reaction mixture was purified by preparative HPLC, and the
Regarding radionuclides for molecular imaging, 125I is gaining
interest as a suitable SPECT agent because it is a readily
available radionuclide with a half-life of 60.2 days and emission
energy in the range of 27−35 KeV. 125I-labeled tracers are
commonly used in molecular biology, and many 125I-labeled
radiopharmaceuticals are commercially available. In addition,
several 125I-labeled imaging probes can be prepared with the aid
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radioactivity was monitored with a γ-counter (see Figure S1,
Supporting Information). The radiolabeling procedure to
obtain 125I-3 resulted in a clean iodination reaction with a
radiochemical yield of 78% for the monoiodinated peptide.
Integrin Binding Assays. The ability of cyclopentapeptide
3 to compete with [125I]echistatin for binding to pure αvβ3
integrin isolated from human placenta was evaluated in solid
phase assays.15 IC50 values showed a binding affinity of
compound 3 toward αvβ3 integrin of 48.0 1.2 nM, slightly
lower than that of peptide 1 (IC50 6.5 2.0 nM) (Figure 2).15
and Ser121/Ser123, whereas the Arg guanidinium group is
responsible for salt bridge interactions with Asp218 and Asp150
side chains. Additional ligand−receptor contacts occur between
Tyr122 in hydrophobic π-stacking and Asn215. The docking
program Autodock 4.0.117 was used to evaluate the binding
energies of selected conformations of ligand 3.18 Docked
conformations were analyzed by taking into account the
binding interactions as observed in the crystal structure of the
bound ligand−protein complex. Docking calculations resulted
in a cluster of conformations characterized by key interactions
with Asp218 and the MIDAS site of the integrin, thus
confirming these interactions as necessary for the molecular
recognition of Arg−Gly−Asp-containing ligands (Figure 3).
Figure 3. RGD ligand 3 (cyan) docked into the binding region of αvβ3
integrin, highlighting protein residues (green) that form key
interactions (dotted lines): Asp218 vs Arg, Ser121 and Mn2+ vs Asp,
Tyr122 and Tyr178 vs D-I-Tyr. Nonpolar hydrogen atoms are omitted
for clarity.
Ligand 3 showed that the Asp side chain is located in the
MIDAS site and interacts with Ser121 and guanidinium group
of Arg, undergoes a salt-bridge interaction with Asp218, and a
monodentate interaction with Asp150. The main conformation
of the cluster showed a different orientation of the D-Tyr
aromatic ring, not facing Tyr-122 in a π-stacking interaction, as
observed for compound 1, but displaying a hydrogen-bond
between the Tyr OH group of the aromatic side chain and the
OH group of Tyr-178 as hydrogen-bond acceptor. The NH
group of D-Tyr showed a hydrogen bond with the Tyr-122 CO
group. Thus, a different binding mode geometry was assessed
for compound 3 as a result of D-Tyr side chain, possibly relating
to a different binding profile with respect to compound 1
(Figure 2).
Cell Biology Assays. Adhesion and Migration of A375 M
Melanoma Cells in the Presence of Morpholine-Based RGD-
Cyclopeptides 1−3. A375 M human melanoma cells express a
wide variety of integrin subunits (αV, β3, β5, α5, β1) and a high
level of αvβ3 heterodimer.12g Figure 4 shows the ability of
ligands 1, 2, and 3, used at final concentrations of 10, 1.0, or 0.1
μM, to inhibit the binding of A375 M melanoma cells to
various RGD-containing substrates, such as vitronectin and
fibronectin. Tests were performed in the presence of 2 mmol/L
MnCl2, in order to switch integrins of tumor cells into the
activated form.6c Figure 4 also shows that ligand 1 inhibits the
binding of melanoma cells to vitronectin to a great extent,
Figure 2. Morpholine-based RGD-cyclopeptides: D-Mor-containing
compound 1, 127I-D-Mor-D-Tyr-containing compound 3. Data are
presented as means SD from three independent experiments, and
error bars represent 95% confidence interval.
Data analyses indicates that compound 3 binds to αvβ3 integrin
according to a one-site binding model (Hill slope −0.92 for
one-site model), whereas cyclopeptide 1 showed binding to
αvβ3 integrin according to a two-site binding model (Hill slope
−0.56 for one-site model), thus suggesting that the iodo-
tyrosine-derived ligand 3 displays a different affinity profile
toward αvβ3 integrin.15
Molecular Modeling. To understand the potential differ-
ences in binding mode of cyclopeptide 3 to αvβ3 integrin, as
compared to ligand 1, a docking simulation was carried out.15
The crystal structure of the complex formed by c[RGDf(Me)V]
and the extracellular fragment of αvβ3 integrin (PDB code:
1L5G) provides a general mode of interaction between the
integrin and its ligands.16 Asp carboxylate and Arg guanidinium
moieties of RGD-based ligands are key structural elements for
receptor recognition. The carboxylate group interacts with the
metal ion-dependent adhesion site (MIDAS) of the Mn2+ ion
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Figure 4. Adhesion of A375 M melanoma cells, HUVECs and dermal fibroblasts to vitronectin, and fibronectin in the presence of compounds 1, 2,
or 3. The values are expressed as % inhibition SEM of cell adhesion relative to control (untreated cells). Significantly different at p < 0.05 as
compared to compound 1.
Figure 5. Radioactivity biodistribution evaluated in a healthy mouse at 60, 120, and 300 min and 24 h after 125I-3 injection.
whereas the inhibition of cell adhesion to fibronectin is less
effective. Ligands 2 and 3 expressed an inhibitory ability on
tumor cell adhesion comparable to that of ligand 1. These
results suggest that ligands 1, 2, and 3 display a quite
comparable affinity for αvβ3 integrin, and the addition of iodine
does not modify the affinity in living cells. A “scratch wound”
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model was used to examine the effect of D-morpholine-
containing RGD-ligands on in vitro migration of melanoma
cells. Tumor cells exposed to ligands 1, 2, and 3 did not modify
their migration rate with respect to nonexposed tumor cells
(control tumor cells). The migration rate (as represented by
the reduction of scratch width) and the morphology of tumor
cells exposed to D-morpholine-containing RGD-ligands were
found comparable at 10 μM, 1 μM, and 10 nM concentration
of the ligand, with respect to control tumor cells (Figure S2,
Supporting Information).
Adhesion and in Vitro Tubulogenesis of HUVEC in the
Presence of Morpholine-Based RGD-Cyclopeptides 1−3. IL-
1β-stimulated HUVECs express integrin subunits (αv, β3, β5, α5,
β1) and αvβ3 heterodimer. Ligands 1, 2, and 3 suppressed the
adhesion of IL-1β-stimulated HUVECs to vitronectin, whereas
they did not completely suppress the adhesion of activated
endothelial cells to fibronectin (Figure 4). We can speculate
that D-morpholine-based RGD-cyclopeptides act preferentially
on the endothelial αvβ3 integrin instead of the α5β1 integrin. To
assess whether ligands 1, 2, and 3 were able to exert an
antiangiogenic activity, we examined the ability of these
peptides to repress in vitro tube formation by HUVEC, seeded
on a Matrigel layer. Figure S3 of Supporting Information shows
that tube formation by HUVEC was not inhibited by D-
morpholine-containing RGD-ligands.
Figure 6. Transaxial slices through mouse chest at implanted
melanoma tumor level (arrows). In upper panels CT images, in
lower panels fused CT and SPECT images. Images were acquired at 40
min after tail vein injection of 125I-3 (250 μCi in 0.2 mL of saline). The
displacement study was achieved with an excess of cold ligand (18 mg/
kg) injected 10 min before the tracer.
cells exposed to ligands 1, 2, and 3 did not modify their
migration rate as compared to nonexposed tumor cells. Several
interactions operate between tumor cells and the extracellular
matrix, thus the inhibition of αvβ3 integrin-mediated cell
adhesion per se might not be sufficient to influence tumor cell
migration.6c,20 Ligands 1, 2, and 3 also repressed the adhesion
of IL-1β-stimulated HUVEC to vitronectin, whereas inhibition
of endothelial cell adhesion on fibronectin was not significant.
At the same time, ligands 1, 2, and 3 did not block in vitro
capillary-like tube formation by HUVEC. We assume that the
reduced affinity of D-morpholine-based RGD-cyclopentapep-
tides for α5β1 integrin fails to influence in vitro angiogenesis.
Indeed, it has been found that only simultaneous inhibition of
both integrins reduces capillary tube formation.11,12h,21
Tumor cells recruit host inflammatory and stromal cells,
which generate a microenvironment able to promote growth
and dissemination of cancer cells.22 Because fibroblasts
represent the most abundant host cells within a tumor
microenvironment,23 we injected a suspension of tumor cells
and αvβ3 integrin expressing HDF into immunodeficient mice
to test in vivo behavior of 125I-3 ligand by micro-SPECT/CT
analysis. We found a high tracer retention within the tumor
mass with a satisfying tumor to background ratio. Co-injection
of 125I-3 and the corresponding unlabeled ligand 3 showed high
selectivity of this ligand for αvβ3 expressing tumor cells.
Thus, the 125I-3 ligand may represent a new radio-
pharmaceutical with the potential of early detection of a
tumor mass and of monitoring the therapeutic response.
Moreover, high affinity of this radiotracer for αvβ3 integrin,
which is characteristic of tumor endothelial cells, can be a
valuable diagnostic tool in a large population of cancers,
including tumors with low level of αvβ3-expressing cells. These
considerations extend the applications of 125I-3 from melanoma
to several tumor histotypes.
Adhesion of HDFs in the Presence of Morpholine-Based
RGD-Cyclopeptides. As shown in Figure 4, ligand 1 suppressed
the adhesion of HDFs to vitronectin and fibronectin with a
high efficiency, whereas ligand 2 and 3 expressed a significantly
reduced efficiency.
Biodistribution and Melanoma Imaging. The radio-
activity distribution was evaluated in a nontumor-bearing
mouse at 60, 120, and 300 min and 24 h post injection (pi)
(Figure 5). The accumulation in gallbladder and kidneys
remained consistent for at least two hours pi, and the tracer was
excreted via renal and hepatobiliary routes, allowing for a fast
clearance of nonspecific bound tracer. Accumulation in the
spleen might be due to the structural similarity of integrin αvβ3
to αIIββ3 integrin expressed on spleen-enriched platelets. The
release of the 125I atom from the molecule and its accumulation
in the thyroid gland increased with time, a phenomenon that
can be avoided by the preadministration of a solution of KI (4
mg/L). At 24 h pi, little radioactivity remained in the organism,
mainly in the thyroid and bowel.
Figure 6 shows the localization of the 125I-3 compound in the
site of the implanted tumor as determined by micro SPECT-
CT imaging. The images clearly show that the 125I-3 ligand has
the capacity to monitor in vivo the growth of αvβ3 integrin-
positive melanoma cells. Binding studies showed a good
displacement of the radiotracer by the parent nonradioactive
compound, with a mean ratio of saturated to displaced
receptors in tumor tissue of 5.753
0.6 and having the
controlateral muscle as the reference tissue.
DISCUSSION AND CONCLUSIONS
■
As reported, αvβ3 integrin-targeted radiotracers are able to
reveal a tumor mass and to monitor growth and diffusion of
tumor cells.19 We tested D-morpholine-based RGD-cyclo-
pentapeptides 1, 2, and 3 in in vitro and in vivo assays, and
they were found to inhibit the adhesion of human melanoma
cells to RGD substrates, such as vitronectin and fibronection.
These ligands proved to be more specific for αvβ3 than α5β1
integrin. “Scratch wound” experiments revealed that melanoma
Finally, the D-Mor-containing cyclopentapeptide ligand 3
might be used in novel therapeutic strategies upon labeling with
131I radioisotope.
EXPERIMENTAL SECTION
General. H-Gly-2-Cl-Trt resin (1.1 mmol/g) was purchased from
Fluka. Chromatographic separations were performed on silica gel
■
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1
Table 1. H and 13C Chemical Shifts of 2 and 3 in DMSO-d6 Solutions at 298 K
1H, 2
1H, 3
13C, 2
13C, 3
H-2
4.28; 3.37
4.59
4.28; 3.34
4.66
C-2
C-3
C-5
C-6
68.8
53.5
66.8
68.2
69.0
53.7
66.4
68.4
H-3
H-5
3.08
3.16; 3.08
3.90; 3.72
8.61
H-6
3.93; 3.70
8.73
Gly NH
Arg NH
Asp NH
D-Tyr NH
Gly H-α
Arg H-α
Arg H-β,γ
Arg H-δ
Asp H-α
Asp H-β
D-Tyr H-α
D-Tyr H-β
D-Tyr H-Ar
7.40
8.56
8.65
8.66
7.19
7.35
4.07
3.99; 3.10
4.01
Gly C-α
44.5
53.3
44.1
53.7
3.99
Arg C-α
1.65; 1.50
3.04
1.63; 1.43
3.06
Arg C-β,γ
Arg C-δ
24.9; 22.0
40.5
24.8; 21.2
40.1
4.78
4.71
Asp C-α
Asp C-β
57.0
56.7
2.82; 2.70
4.47
2.88; 2.56
4.51
39.6
39.8
D-Tyr C-α
D-Tyr C-β
D-Tyr C-Ar
49.4
50.4
2.14
2.32
28.7
35.8
6.95; 6.60
7.66; 7.34; 7.24
115.3; 130.1
115.8; 129.8; 139.1
(Kieselgel 60, Merck) using flash-column techniques. Rf values refer to
TLC carried out on 25 mm silica gel plates (Merck F254) with same
eluant as for column chromatography. Solid-phase reactions were
carried out on a shaker using solvents of HPLC quality. Bromophenol
Blue (BB) test was performed as follows: a small amount of resin
beads were suspended in a vial with 0.5 mL of DMF, two drops of a
1% solution of bromophenol blue in dimethylacetamide were added,
and the sample was observed. The BB test was considered positive
(presence of free amino groups) when the resin beads turned blue and
negative (absence of free amino groups) when the beads remained
colorless. ESI mass spectra were carried out on a ion-trap double
quadrupole mass spectrometer using electrospray (ES+) ionization
techniques, and a normalized collision energy within the range of 25−
32 eV for MSMS experiments. Ligands 2 and 3 were purified by
Beckman-Gold HPLC system equipped with a reverse-phase column
(Alltima C18 10 μm, 250 mm × 10 mm, Alltech) using H2O/CH3CN
gradient eluant buffered with 0.1% TFA (flow rate 2.5 mL/min, λ 254
nm, gradient eluant: CH3CN 10%/5 min, CH3CN 10−90%/25 min).
Analytical HPLC analyses were performed on Dionex Ulltimate 3000
system equipped with a reverse-phase column (Alltima C18 5 μm, 250
mm × 4.6 mm, Alltech) and the same gradient eluant. Ligand 125I-3
was purified by Beckman-Gold HPLC system equipped with a reverse-
phase column (C-8 Waters Spherisorb S5 250 mm × 4.6 mm) and the
same gradient eluant. Na125I in 0.2N NaOH solution was purchased
from PerkinElmer (10 μL, 100 mCi/mL, 17 Ci (629GBq)/mg, 125I
97%, 126I < 3%, radiochemical purity >99%, carrier free). HPLC
analysis was used to determine purity and all tested compounds
possessed >95% purity.
c[RGDy-(3R)-carboxymorpholine] (2). H-Gly-2-Cl-Trt resin
(466 mg, 0.5 mmol) was used as starting material. Fmoc-deprotections
and completion of each coupling reaction were assessed by performing
a BB test. Coupling was performed with a solution consisting of N-
Fmoc-Arg(Pbf)-OH (973 mg, 1.5 mmol, 3 equiv), HOBt (203 mg, 1.5
mmol, 3 equiv) in DMF (3 mL), and DIC (203 μL, 1.5 mmol, 3
equiv) was added dropwise, at 0 °C. The resulting mixture was stirred
for 10 min at 0 °C and for a further 10 min at room temperature and
then was added to the resin. This mixture was shaken at room
temperature for 24 h. The solution was drained, and the resin was
sequentially washed with 5% DIPEA in DMF (3 × 5 mL) and DMF (5
× 5 mL). Fmoc deprotection was performed with 30% piperidine in
DMF (10 mL) for 30 min, followed by resin washings with DMF (3 ×
10 mL). After deprotection, H-Arg(Pbf)-Gly-2-Cl-Trt resin was
suspended in a solution of HOBt (356.8 mg, 2.64 mmol, 6 equiv),
DIC (415.6 μL, 2.64 mmol, 6 equiv), and N-Fmoc-(R)-morpholine-3-
carboxylic acid (465 mg, 1.32 mmol, 2.64 equiv) in DMF (3 mL) and
shaken for two days. The solution was drained, and resin was washed
with 5% DIPEA in DMF (3 × 5 mL) and DMF (5 × 5 mL). After
deprotection, H-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin
was suspended in a solution of HOBt (405 mg, 3 mmol, 6 equiv), DIC
(472 μL, 3 mmol, 6 equiv), and N-Fmoc-D-Tyr(t-Bu)-OH (689 mg,
1.5 mmol, 3 equiv) in DMF (3 mL) and shaken for three days. The
solution was drained and the resin washed with 5% DIPEA in DMF (3
× 5 mL) and DMF (5 × 5 mL). After deprotection, H-D-Tyr(t-Bu)-
(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-Cl-Trt resin was suspended
in a solution of TBTU (802.7 mg, 2.5 mmol, 5 equiv), N-Fmoc-Asp(t-
Bu)-OH (1.028 g, 2.5 mmol, 5 equiv), and DIPEA (428 μL, 2.5 mmol,
5 equiv) in DMF (3 mL) and shaken for three days. The solution was
drained and resin washed with 5% DIPEA in DMF (3 × 5 mL) and
DMF (5 × 5 mL). N-Fmoc-Asp(t-Bu)-D-Tyr(t-Bu)-(3R)-carboxymor-
pholine-Arg(Pbf)-Gly-2-Cl-Trt resin was deprotected and washed with
DMF (5 × 5 mL) and DCM (5 × 5 mL). In a solid-phase reaction
vessel H-Asp(t-Bu)-D-Phe-(3R)-carboxymorpholine-Arg(Pbf)-Gly-2-
Cl-Trt resin was treated with 5 mL of 1% TFA/DCM solution (10
× 2 min). The filtrates were immediately neutralized with a 10%
pyridine/MeOH solution (1 mL), and then the resin was washed with
DCM (3 × 5 mL). Fractions (TLC eluant 4:1 DCM/MeOH) were
combined and concentrated under reduced pressure to yield a residue,
which was suspended in H2O and purified from the pyridinium salts by
size-exclusion chromatography (AMBERLITE XAD2 resin, H2O then
MeOH). Evaporation of combined MeOH fractions containing the
product afforded the side-chain protected compound (493 mg, 33%)
as a white solid material. The linear peptide (493 mg, 0.5 mmol) was
dissolved under a nitrogen atmosphere in DMF (120 mL), HATU
(152.1 mg, 0.4 mmol, 0.8 equiv), and TBTU (354 mg, 1.1 mmol, 2.2
equiv) and DIPEA (257 μL, 1.5 mmol, 3 equiv) were added. The
resulting mixture was stirred for two days at room temperature. The
solvent was distilled off under reduced pressure, and the residue was
dissolved in H2O (60 mL) and extracted with EtOAc (3 × 80 mL).
The organic phase was washed twice with 5% NaHCO3, dried with
Na2SO4, and evaporated under reduced pressure. The crude residue
was purified by flash column chromatography (9:1 DCM−MeOH, Rf
0.37) to give the protected cyclic peptide (140 mg, 30%) as a yellow
solid material. HPLC: tR = 24.30, purity 97%. Side chain protected
cyclic peptide (140 mg, 0.15 mmol), was treated with 95:2.5:2.5 TFA/
H2O/TIS mixture (20 mL) for 2 h. The reaction mixture was
evaporated under reduced pressure, and the residue was dissolved in
i
H2O (60 mL). The aqueous phase was washed with Pr2O (3 × 60
mL) and freeze-dried. The crude residue was purified by semi-
preparative HPLC to give the side-chain deprotected 2 (30 mg, 34%),
as a white solid. Trifluoroacetate ion was replaced with chloride ion by
ion-exchange chromatography (AMBERLITE IRA-96 resin, chloride
form). See Table 1 for 1H and 13C NMR data. ESI-MS m/z 605.14 (M
+ H+, 100). ESI-MSMS m/z 605.5 (M + H+, 1), 588.0 (100), 577.0
(30), 570.0 (17), 559.9 (42), 546.0 (11), 533 (12), 520.0 (72). Anal.
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Calcd for C26H37ClN8O9: C, 48.71; H, 5.82; N, 17.48. Found: C,
48.88; H, 5.91; N, 17.32. HPLC: tR = 12.1, 95% purity.
humidified incubator containing 10% CO2. Then 5.0 × 105 melanoma
cells or 1 × 106 HDF were seeded in 100 mm dishes and propagated
every 3 days by incubation with a trypsin−EDTA solution. Cultures
were periodically monitored for mycoplasma contamination. Primary
cultures of human umbilical vein endothelial cells (HUVEC) were
obtained from Lonza (USA). HUVEC grown in endothelial cell
growth medium-2 (EGM-2) containing endothelial cell growth
supplements were subcultivated using a trypsin-EDTA solution, 1:3
split ratio. Cells between passages 2 and 4 grown to confluence in
plates coated with 1% bovine gelatin (Sigma, St. Louis) were used in
the experiments.
RNA Isolation and Polymerase Chain Reaction (PCR). Total
RNA extracted using RNAgents (Total RNA Isolation System,
Promega, Madison, WI) was determined spectrophotometrically.
Complementary DNA (cDNA) was synthesized from 2 μg of total
RNA using 1 μL of ImProm-II reverse transcriptase (Promega).
Aliquots of 2 μL of the cDNA were used for PCR amplification. The
specific primers used for the identification of human αv, α5, β1, β3, β5,
and GAPDH are reported in Table S1 of Supporting Informations. All
PCR experiments were conducted using 0.05 U/μL of Go-Taq
Polymerase (Promega). Amplification was carried out on a Perkin-
Elmer Thermal cycler. Then 8 μL of each PCR products were
visualized after electrophoresis in a 2% agarose gel containing 0.5 μg/
mL of ethidium bromide. cDNA products were evaluated on the basis
of a standard PCR marker.
Flow Cytometry Assay. Tumor cells were detached by gentle
treatment with Accutase, a 0.5 mM EDTA solution, washed, and
incubated for 1 h at 4 °C in the presence of anti αvβ3 monoclonal
antibody (1 μg/50 μL, Anti-Integrin αvβ3, clone LM609, Millipore).
Cells were then washed and incubated for 1 h at 4 °C with a secondary
antibody, 5 μg/mL goat antimouse IgG conjugated with FITC (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA). αvβ3 -positive cells were
analyzed at 488 nm on the flow cytometer FACScan system (BD-
FACS Canto).
Cell Adhesion Assay. Plates (96 wells) were coated with
fibronectin (1 μg/mL) or vitronectin (10 μg/mL) by overnight
incubation at 4 °C and then incubated at 37 °C for 1 h with 1% BSA in
PBS. Melanoma cells, IL-1β (400 U/mL)-stimulated HUVECs, or
human dermal fibroblasts (HDF) were resuspended in serum-free
medium and exposed to ligands 1, 2, or 3 (final concentration was 0.1,
1, or 10 μM) at 37 °C for 30 min to allow for ligand−integrin
equilibrium to be reached. Melanoma cell adhesion assays were
performed in the presence or in the absence of 2 mmol/L MnCl2.
Cells were plated (4−5 × 104 cells/well) and incubated at 37 °C for 2
h. Nonadherent cells were removed with PBS, and adherent cells were
stained with 0.5% crystal violet solution in 20% methanol. After 2 h of
incubation at 4 °C, plates were examined at 540 nm in a counter
ELX800 (Biotek Instruments). Experiments were conducted in
triplicate and repeated at least three times. Data are presented as
means SD from three independent experiments.
c[RGD-(3′I-y)-(3R)-carboxymorpholine] (3). A solution of
ligand 2 (12.0 mg, 0.020 mmol) in PBS (2 mL) was added to sodium
iodide (3.07 mg, 0.020 mmol), followed by chloramine-T hydrate
(4.66 mg, 0.020 mmol). The reaction mixture was stirred at room
temperature for 20 min and then quenched with a solution of 5%
sodium metabisulphite (0.8 mL). The mixture was extracted with
DCM (3 × 5 mL) and loaded on a preconditioned C18 Sep-pak
column. The C18 column was washed with water (6 mL) and
subsequently eluted with MeOH. The crude product was purified by
semipreparative HPLC to obtain ligand 3 (4.67 mg, 32%). See Table 1
1
for H and 13C NMR data. ESI-MS m/z 731.25 (M + H+, 100). ESI-
MSMS m/z 731.2 (M + H+, 15), 714.1 (100), 685.9 (39), 672.1 (22),
646.1 (76). Anal. Calcd for C26H36ClIN8O9: C, 40.72; H, 4.73; N,
14.61. Found: C, 40.81; H, 4.83; N, 14.12. HPLC: tR = 15.2, 96%
purity.
c[RGD-(3′125I-y)-(3R)-carboxymorpholine] (125I-3). A solution
of PBS (100 μL, 0.05 M) containing Na125I (20 μL, 2 mCi, 0.918 nM),
peptide 2 (10 μL of a 0.05 M PBS solution, 16.5 nmol), and
chloramine-T (1.88 μL of a freshly prepared 0.05 M PBS solution, 8.24
nmol, 9 equiv) was prepared. The reaction mixture was stirred at room
temperature for 20 min and then quenched by adding a solution of
sodium metabisulphite (2.18 μL of a freshly prepared 0.05 M PBS
solution, 8.24 nmol, 9 equiv). The mixture was extracted with DCM (3
× 0.5 mL) and purified by HPLC, and the radioactivity of fractions
collected every 30 s was determined with a γ-counter (Supporting
Information Figure S1). Fractions containing the chemically pure 125I-
3 were pooled together (1.55 mCi, 78% radiochemical yield) and used
for in vivo experiments after solvent removal. HPLC: tR = 15.2, 98%
radiochemical purity, >99% chemical purity.
Solid-Phase Integrin Binding Assay. [125I]-Echistatin, labeled by
the lactoperoxidase method24 to a specific activity of 2000 Ci/mmol,
was purchased from GE Healthcare, and integrin αvβ3 from human
placenta was purchased from Chemicon International Inc., Temecula,
CA.
Purified αvβ3 integrin was diluted in coating buffer (20 mM Tris
(pH 7.4), 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2)
at concentrations of 500 or 1000 ng/mL. An aliquot of diluted integrin
(100 μL/well) was added to a 96-well microtiter plate (Optiplate-96
HB, PerkinElmer Life Sciences, Boston, MA), and plates were
incubated overnight at 4 °C. Plates were washed once with
blocking/binding buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 2
mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 1% BSA) and incubated at
room temperature for additional 2 h. Plates were rinsed twice with
same buffer, and then competition binding assays were performed with
a constant concentration of [125I]-Echistatin (0.05 nM). Concen-
trations of tested compound ranged from 0.01 to 100 nM. All assays
were performed in triplicate in a final volume of 0.2 mL, each
containing the following species: 0.05 mL of [125I]-Echistatin, 0.04 mL
of tested compound, and 0.11 mL of blocking/binding buffer.
Nonspecific binding was defined as [125I]-Echistatin bound in presence
of an excess (1 μM) of unlabeled echistatin. After incubation at room
temperature for 3 h, plates were washed three times with blocking/
binding buffer and counted in a Top-Count NXT microplate
scintillation counter (PerkinElmer Life Sciences, Boston, MA) using
200 μL/well of MicroScint-40 liquid scintillation (PerkinElmer Life
Sciences, Boston, MA).
Animal Model and Study Design. In vivo experiments were
conducted in accordance with national guidelines and approved by the
Ethical Committee of the Animal Welfare Office of the Italian Work
Ministry and conforming to legal mandates and Italian guidelines for
care and maintenance of laboratory animals. Eighteen-week-old SCID
bg/bg mice (Charles River Laboratories International) were injected
subcutaneously in the shoulder area with a suspension of melanoma
cells and 20 ng/mL TGFβ-activated fibroblasts. Then 1 ×106 A375 M
melanoma cells and 0.5 × 106 HDF were previously resuspended in
100 μL of PBS diluted 1:1 with a Matrigel solution (BD Biosciences).
Animals were kept under pathogen-free conditions and allowed to feed
freely. Tumor growth was monitored daily.
Data are shown as means
SD from three independent
experiments. IC50 values were determined by fitting binding inhibition
data by nonlinear regression using GraphPad Prism 4.0 Software
Package (GraphPad Prism, San Diego, CA).
Cell Lines and Culture Conditions. The A375 M human
melanoma cell line was obtained from the American Type Culture
Collection (ATCC, Rockville, MD).12g Human dermal skin fibroblasts
(HDF) were kindly donated by Dr. Daniela Monti, Department of
Experimental Pathology and Oncology, University of Florence.
Melanoma cells and HDF were grown in Dulbecco’s Modified Eagle
Medium, containing 4500 mg/L glucose (DMEM 4500, GIBCO)
supplemented with 10% fetal calf serum (FCS) at 37 °C in a
Small-Animal SPECT and CT Studies. In vivo studies were
conducted using a “hybrid” imaging system for small laboratory
animals allowing for the study of biological processes using minimally
invasive procedures. Engrafted tumor tracer uptake was studied at
different time-points after tail vein injection. Binding specificity to
integrin receptors was evaluated by in vivo displacement studies, in
which a high dose of cold tracer was injected 10 min before
radiolabeled one. Imaging studies were performed using the Flex
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Triumph preclinical imaging system, which combines positron (PET)
and single-photon emission (SPECT) tomography with the emission
tomography (CT) for anatomic coregistration (GE Healthcare/
Gamma Medica-Ideas, Waukesha, WI). 125I-iodinated tracers 125I-3
was studied in vivo by SPECT, allowing for use directly the in vitro
tracer data to the in vivo experimentation. Imaging studies required
monitoring of anesthetized animals throughout the imaging procedure,
and anesthesia was conducted in accordance with National Guidelines
and approved by the Ethical Committee. Given that anesthesia in
general causes hypothermia in small animals, appropriate warming was
provided during imaging procedure by using a heating bed set at 37
°C. Physiological monitoring was achieved by measuring respiration
rate with a pressure pad placed under the mouse abdomen. Saturation
experiments (SE) to visualize tumors and displacement experiments
(DE) were performed. In SE, 125I-3 (7−10 MBq in 200 μL of
physiological saline) was injected via tail vein into tumor bearing
animals kept under anesthesia. After 30 min, a CT aquisition was
performed, followed by SPECT scan, starting at 40 min pi. CT scan
was acquired with 50 kV and 320 μA tube settings. The magnification
factor was set to 2, resulting in an axial field of view of 59.2 mm. 512
projections, 500 μs each, were acquired on a circular orbit in 256 s
with an acquisition matrix of 1184 × 1120, 100 μm, pixel. SPECT
projections were acquired with a 5-pinhole high-resolution collimator
(1 mm hole diameter, 75 mm focal distance) with radius of rotation of
40 mm resulting in an axial field of view of 52.9 mm. then 64
projections, 20 s each, were acquired on a circular orbit for a total
acquisition time of about 22 min. Acquisition matrix was 80 × 80, 1.5
mm pixel size. Energy window was set on the 125I photopeak ranging
from 20 to 36 KeV. The DE were performed by injecting 18 mg/kg of
compound 3 10 min before injection of tracer. Once scanning was
complete, the animals were recovered in a housing unit and monitored
until awake.
Data Analysis of Small-Animal SPECT and CT Studies. CT
image data set was reconstructed using a cone-beam FBP algorithm
(FDK) and an optimum noise filter. Reconstruction matrix was 512 ×
512 × 512, 180 μm pixel size. SPECT images were reconstructed with
ordered subset expectation maximization algorithm (8 subsets and 5
iterations) and a reconstruction matrix of 60 × 60 × 60, 1.5 mm pixel
size. No correction for radiation attenuation and scatter was applied.
Fused images were visually analyzed to detect the engrafted tumor
mass, and regional SPECT activities were measured using the analysis
software Vivid (Gamma Medica-Ideas, Northridge, CA) by delineating
the region of interest (ROI) of tumor in fused CT images. A similar
ROI was generated for muscle tissue on contralateral side of tumor.
Tumor ROI activity was normalized (nTA) with the following
formula:
Cluster analysis was performed on docked results using a root-mean-
square (rms) tolerance of 1.5 Å. Analysis of binding mode, calculation
of binding energy, and prediction of binding activity of docked
conformations were carried out using PyMol Autodock Tools plugin
within PyMol software.26
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental methods, radiochromatogram of 125I-3,
and additional cell biology assays data. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For A.G.: phone, +39 055 4573481; fax, +39 055 4573569; E-
mail, antonio.guarna@unifi.it. For A.P.: phone, +39 055
7947527; fax, +39 055 4224392; E-mail, a.pupi@dfc.unifi.it.
For L.C.: phone, +39 055 4598207; fax, +39 055 4598900; E-
mail, lido.calorini@unifi.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work has received financial support from the Istituto
Toscano Tumori and MIUR (PRIN08, Grant
2008J4YNJY_003). Also, financial contributions from CINM-
PIS, University of Florence, and Fondazione Roma are
acknowledged. Dr. Alessandro Sisti is acknowledged for
carrying out preliminary labeling experiments. Ente Cassa di
Risparmio is acknowledged for the granting the MicroPET/
SPECT/CT instrument.
ABBREVIATIONS USED
■
SPECT, single positron emission computed tomography; PET,
positon emission tomography; RGD, Arg-Gly-Asp; VGP,
vertical growth phase; CT, computed tomography; VN,
vitronectin; FN, fibronectin; HUVEC, human umbilical vein
endothelial cells; IL-1β, interleukin-1β; ESI-MSMS, electropray
ionization tandem mass spectrometry; EGM-2, endothelial cell
growth medium-2; EDTA, ethylenediaminetetraacetic acid;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FACS,
fluorescence-activated cell sorting; FITC, fluorescein isothio-
cyanate; PBS, phosphate buffered saline; HUVEC, human
umbilical vein endothelial cells; HDF, human dermal skin
fibroblasts; EMT, epithelial-mesenchymal transition; TGF,
transform grown factor; pi, post injection
TA − BA
nTA =
BA
where TA is tumor activity and BA is background activity measured in
muscle tissue contralateral to tumor.
Docking Calculations. Automated docking studies were carried
out by Autodock 4.0.1 program,17 using the Lamarckian Genetic
Algorithm (LGA) as a search engine. The AutoDockTools 1.4.5
(ADT) graphical interface25 was used to prepare integrin and ligands
PDBQT files. Coordinates of ligand 3 were retrieved from lowest
energy conformers resulting from molecular mechanics calculations
starting from geometry of ligand 1, whereas coordinates of αvβ3
receptor were retrieved from the Protein Data Bank (PDB code:
1L5G), and ligand−protein complex was unmerged for achieving free
receptor structure. Water molecules were removed. For protein
receptor and ligand 3, all hydrogens were added, Gasteiger charges
were computed, and nonpolar hydrogens were merged. A charge value
of +2.0 to each Mn atom of protein receptor was successively added.
Three-dimensional energy scoring grids of 0.375 Å resolution and 40
Å × 40 Å × 40 Å dimensions were computed. Center of grid was set to
be coincident with mass center of ligands preliminary fitted on the X-
ray structure of c[RGDf(Me)V] in the αvβ3 complex (1L5G). A total
of 50 runs with a maximum of 2500000 energy evaluations were
carried out for each ligand, using the default parameters for LGA.
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