Radiolabeled γ-AApeptides: A new class of tracers for positron emission tomography

Article abstract of DOI:10.1039/c2cc33620k

A γ-AApeptide-based tracer for positron emission tomography imaging of integrin α_vβ₃ is reported. Despite its shorter sequence and linear nature, this tracer had comparable integrin α_vβ₃ binding affinity to the cyclic arginine-glycine-aspartic acid peptide but significantly higher resistance to enzymatic degradation and better stability. This journal is

Full text of DOI:10.1039/c2cc33620k

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Radiolabeled c-AApeptides: a new class of tracers for positron emission
tomographyw
a
b
Yunan Yang,z Youhong Niu,z Hao Hong,a Haifan Wu,b Yin Zhang,a Jonathan W. Engle,a
Todd E. Barnhart,a Jianfeng Cai*b and Weibo Cai*a
Received 7th March 2012, Accepted 10th June 2012
DOI: 10.1039/c2cc33620k
A c-AApeptide-based tracer for positron emission tomography
imaging of integrin avb3 is reported. Despite its shorter sequence
and linear nature, this tracer had comparable integrin avb3
binding affinity to the cyclic arginine-glycine-aspartic acid peptide
but significantly higher resistance to enzymatic degradation and
better stability.
for PET imaging,7 which opened the door to a fertile area of
future research and development.
We recently designed a new class of peptidomimetics that
are termed ‘‘g-AApeptides’’,8 which have shown promising
potential for various biological applications such as effective
disruption of protein–protein interactions,8 recognition of
specific nucleic acids,9 and as novel antimicrobial agents.10 In
addition, these g-AApeptides are resistant to proteolytic degra-
dation and are amenable to limitless diversification. Since
g-AApeptides project the same number of side chains as that
of peptides of the same length, they are expected to be ideal
candidates for short peptide mimicry. To further explore their
potential applications in biomedical research, and to develop
novel molecular imaging agents, herein we report short and
linear g-AApeptide-based RGD mimetics (Fig. 1), which can be
employed for in vivo PET imaging of integrin avb3 expression.
Our results demonstrate that this new class of RGD mimetics is
comparable to the commonly used c(RGDyK) (where y denotes
D-tyrosine) peptide in terms of integrin avb3 binding affinity and
specificity. Furthermore, they are much more protease-resistant,
hence are more suitable for PET imaging applications.
Positron emission tomography (PET) has been widely used in
clinical oncology for tumor diagnosis, staging, and treatment
monitoring.1 Development and clinical translation of novel
molecularly targeted PET tracers will facilitate future person-
alized medicine of cancer patients, such as patient stratification
and monitoring the therapeutic responses to anti-cancer drugs.2
Non-invasive PET imaging of tumor angiogenesis (i.e. new
blood vessel formation) has gained tremendous interest over
the last decade,3 since the development and metastasis of all
solid tumors depend on tumor angiogenesis.4 Among the
many proteins that are involved in tumor angiogenesis, integrin
avb3 is one of the most intensively studied and several PET
tracers targeting this cell adhesion molecule have entered
clinical investigation.5 Frequently overexpressed on the tumor
neovasculature, as well as cancer cells of many tumor types
(e.g. lung/prostate/breast cancer and glioblastoma), integrin avb3
is an attractive target for both cancer diagnosis and therapy.6
Since integrin avb3 binds tightly to extracellular matrix
proteins that contain the Arg-Gly-Asp (RGD) tripeptide
epitopes, a wide variety of peptides/peptidomimetics based
on the RGD motif have been investigated for anti-cancer drug
development and/or cancer imaging.5a,6 The Achilles’ heel of
peptides is that they are susceptible to degradation by a variety
of enzymes, hence are not very stable in vivo. Many approaches
have been employed to improve the in vivo stability of peptide-
based imaging/therapeutic agents, such as cyclization and the
use of unnatural amino acids.5a Recently, a PET tracer based
on peptoids (i.e. poly-N-substituted glycines) was reported
g-AA1 is a g-AApeptide designed to mimic the tripeptide
RGD. An additional phenyl moiety was included in the molecule
to provide a balance between hydrophilicity and hydrophobicity,
a Departments of Radiology and Medical Physics, University of
Wisconsin - Madison, WI 53705, USA. E-mail: wcai@uwhealth.org
b Department of Chemistry, University of South Florida,
4202 E. Fowler Ave, Tampa, FL 33620, USA.
E-mail: jianfengcai@usf.edu
Fig. 1 The RGD tripeptide and the g-AApeptides. g-AA1 is a
g-AApeptide that mimics RGD. g-AA2 is a DOTA conjugated g-AA1
for 64Cu-labeling and PET imaging.
w Electronic supplementary information (ESI) available: Synthesis,
characterization, and experimental details. See DOI: 10.1039/c2cc33620k
z Yunan Yang and Youhong Niu contributed equally to this work.
c
7850 Chem. Commun., 2012, 48, 7850–7852
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uptake in the U87MG cells as FITC-c(RGDyK), as evidenced
by flow cytometry (Fig. 3a). Blocking the receptor with 2 mM
of unconjugated c(RGDyK) significantly reduced the uptake of
both FITC-g-AA1 and FITC-c(RGDyK) to a similar extent.
U87MG cell binding assay using 64Cu-DOTA-c(RGDyK) as the
radioligand revealed that the IC50 values were 831 and 897 nM
for g-AA1 and g-AA2, respectively (Fig. 3b). These values are
slightly lower but comparable to that of c(RGDyK), with an
IC50 value of 639 nM in the same assay. Together, these findings
indicated that g-AA1/g-AA2 and the c(RGDyK) peptide have
similar binding affinity and specificity to integrin avb3 in vitro.
To enable 64Cu-labeling and PET imaging, DOTA (1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid) was linked to
g-AA1 via a 6-aminohexanoic acid linker, which was termed
g-AA2 (i.e. DOTA-g-AA1, Fig. 1). 64Cu-labeling of g-AA2,
including final purification with HPLC, took 90 ꢁ 15 min
(n = 8). The decay-corrected radiochemical yield was
50 ꢁ 15%, based on 2 mg of g-AA2 per 37 MBq of 64Cu, with
a radiochemical purity of >95%. The specific activity of
64Cu-g-AA2 was measured to be B9 GBq mgꢀ1 of g-AA2.
The c(RGDyK) peptide was conjugated with DOTA, purified
by HPLC, and labeled with 64Cu in a similar manner.
Fig. 2 The superimposition of energy-minimized g-AA1 (green) and
c(RGDyK) (cyan). The energy minimization and superimposition was
carried out using the ChemBioOffice program.
as was found in many RGD-containing peptides for imaging
and/or therapeutic applications,6 which is not expected to interfere
with integrin avb3 binding. To rationalize such design, structural
studies were carried out by superimposing the energy-minimized
structure of g-AA1 onto that of c(RGDyK). As shown in Fig. 2,
the guanidino and carboxyl groups within g-AA1 superimpose
very well with the functional groups of Arg and Asp residues from
c(RGDyK), which are responsible for the recognition of integrin
avb3. Thus, computer modeling supports the straightforward and
effective design of g-AApeptides for RGD motif mimicry. Future
systematic studies will be carried out to investigate the effect
of different functional groups on integrin avb3 binding of the
g-AApeptide-based RGD mimetics.
To further evaluate the capability of the g-AApeptides for
RGD mimicry, g-AA1 and the c(RGDyK) peptide, an extensively
studied and validated high affinity antagonist for integrin avb3,5a
were each conjugated to FITC. After purification by high perfor-
mance liquid chromatography (HPLC), FITC-g-AA1 and FITC-
c(RGDyK) were compared for integrin avb3 binding affinity and
specificity in U87MG human glioblastoma cells that express a
high level of integrin avb3.11 At a 5 mg mLꢀ1 concentration which
is under non-saturating conditions (i.e. the fluorescence signal was
in the 102–103 range instead of 104), FITC-g-AA1 has similar
Before PET imaging was carried out to evaluate the in vivo
behaviour of 64Cu-labeled g-AA2, enzymatic stability of the tracer
was investigated. Pronase, a mixture of proteinases isolated from
the extracellular fluid of Streptomyces griseus, was used to
compare the enzymatic stability of the two PET tracers.8,12 After
incubation with 0.1 mg mLꢀ1 of pronase at 37 1C in 100 mM
ammonium bicarbonate buffer (pH 7.8) for various time periods,
stability of the two tracers was compared using radio-HPLC.
64Cu-g-AA2 exhibited markedly better stability than
64Cu-DOTA-c(RGDyK) (Fig. 4). The stability of c(RGDyK)
is expected to be significantly higher than the natural RGD
peptide, attributed to the inclusion of a D-Tyr residue and head-
to-tail cyclization, both of which are proven strategies for
improving the stability of natural peptides. However, B8% of
64Cu-DOTA-c(RGDyK) was already degraded at 0.5 h post-
treatment, with 100% enzyme degradation at 2 h and 8 h post-
treatment. In comparison, 64Cu-g-AA2 only had 4%, 8%, and
15% degradation at 0.5 h, 2 h, and 8 h post-treatment,
respectively. The UV traces were similar to the radio-HPLC
results (see ESIw). The fact that 64Cu-g-AA2 is much more
enzymatically stable than 64Cu-DOTA-c(RGDyK) makes
g-AApeptides a promising class of targeting ligands for PET
imaging applications, which possess exceptional stability.
After demonstrating the excellent stability of 64Cu-g-AA2
in vitro, serial in vivo PET imaging was carried out in the U87MG
Fig. 3 (a) Flow cytometry analysis of FITC-conjugated g-AA1 and
c(RGDyK) peptide in U87MG cells at a 5 mg mLꢀ1 concentration.
Blocking experiments with 2 mM of c(RGDyK) peptide were also
performed to confirm the specificity for integrin avb3. (b) U87MG cell
binding assay demonstrated that both g-AA1 and g-AA2 bind to
integrin avb3, similar to the c(RGDyK) peptide.
Fig. 4 Serial radio-HPLC profiles of 64Cu-DOTA-c(RGDyK) and
64Cu-g-AA2 before and after incubation in pronase at 37 1C.
c
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24 h p.i. respectively (n = 3). Since 64Cu-g-AA2 undergoes
both hepatobiliary and renal clearance, tracer uptake was also
observed in the liver/kidneys.
Administering a blocking dose of the c(RGDyK) peptide
reduced the U87MG tumor uptake significantly to 0.5 ꢁ 0.1,
0.4 ꢁ 0.1, 0.4 ꢁ 0.1, and 0.4 ꢁ 0.1%ID gꢀ1 at 0.5, 2, 4, and
24 h p.i., respectively (n = 3; P o 0.05 at 0.5, 2, and 4 h p.i.
when compared with mice injected with 64Cu-g-AA2 only;
Fig. 5a–c), which demonstrated integrin avb3 specificity of the
tracer in vivo. Liver uptake of 64Cu-g-AA2 was higher in the
blocking group, being 7.2 ꢁ 2.0, 6.1 ꢁ 1.6, 5.3 ꢁ 1.3, and
4.0 ꢁ 0.6%ID gꢀ1 at 0.5, 2, 4, and 24 h p.i. respectively (n = 3).
Radioactivity in the blood (0.5 ꢁ 0.1, 0.4 ꢁ 0.1, 0.4 ꢁ 0.1, and
0.4 ꢁ 0.1%ID gꢀ1 at 0.5, 2, 4, and 24 h p.i., respectively; n = 3)
was lower for the blocking group at early time points, indicating
faster blood clearance of the tracer (Fig. 5b). After the last PET
scans, all mice were euthanized for biodistribution studies to
validate the PET data. The %ID gꢀ1 values of 64Cu-g-AA2 in
the tumor and major organs obtained from biodistribution
studies (Fig. 5d) matched well with the results from ROI
analysis of the PET scans, confirming that PET can enable
accurate quantification of tracer distribution in vivo.
In summary, the 64Cu-labeled g-AApeptide-based RGD
mimetic exhibited comparable integrin avb3 binding affinity
to the c(RGDyK) peptide but significantly higher resistance to
enzymatic degradation and better in vivo stability, despite its
shorter sequence and linear nature. Integrin avb3 specificity,
fast blood clearance, and good tumor contrast of 64Cu-g-AA2
established g-AApeptides as a novel class of enzymatically
stable targeting ligands for molecular imaging applications.
This work was supported in part by W81XWH-11-1-0644,
W81XWH-11-1-0648, the Elsa U. Pardee Foundation, and
UW/USF startup funds.
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7852 Chem. Commun., 2012, 48, 7850–7852
This journal is The Royal Society of Chemistry 2012