Radiolabeled antagonistic bombesin peptidomimetics for tumor targeting

Article abstract of DOI:10.1002/jlcr.3162

The replacement of amide bonds in the backbone of peptides by proteolytically stable 1,2,3-triazole isosteres can provide novel peptidomimetics with promising properties for the development of tumor-targeting radiopeptides. On the basis of our previous work with radiolabeled agonistic bombesin (BBN) derivatives of the sequence [Nle¹⁴]BBN(7-14), we substituted selected amide bonds of the structurally closely related antagonistic peptide analog JMV594. With the exception of the C-terminal modification, amide-to-triazole substitutions tolerated by [Nle ¹⁴]BBN(7-14) without loss of biological function led to abolished receptor affinity in the case of JMV594. These findings provide an additional piece of evidence for the currently disputed differences in the modes of action of agonistic and antagonistic gastrin-releasing peptide receptor (GRPR)-targeting radiopeptides.

Full text of DOI:10.1002/jlcr.3162

Special Issue Article
Received 1 August 2013,
Accepted 29 October 2013
Published online 11 December 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3162
Radiolabeled antagonistic bombesin
peptidomimetics for tumor targeting†‡
*
Ibai E. Valverde, Elena Huxol, and Thomas L. Mindt
The replacement of amide bonds in the backbone of peptides by proteolytically stable 1,2,3-triazole isosteres can provide
novel peptidomimetics with promising properties for the development of tumor-targeting radiopeptides. On the basis of
our previous work with radiolabeled agonistic bombesin (BBN) derivatives of the sequence [Nle14]BBN(7–14), we substituted
selected amide bonds of the structurally closely related antagonistic peptide analog JMV594. With the exception of the
C-terminal modification, amide-to-triazole substitutions tolerated by [Nle14]BBN(7–14) without loss of biological function
led to abolished receptor affinity in the case of JMV594. These findings provide an additional piece of evidence for the
currently disputed differences in the modes of action of agonistic and antagonistic gastrin-releasing peptide receptor
(GRPR)-targeting radiopeptides.
Keywords: 1,2,3-triazoles; amide bond mimics; bombesin; antagonists; radiopeptides; peptidomimetics; click chemistry
antagonists has not yet been fully elucidated, they represent
Introduction
an interesting new class of tumor-targeting peptidic vectors for
the development of radiotracers.6
The development of radiopharmaceuticals based on peptides as
tumor-targeting moieties (vectors) has been a focus of cancer
We have recently shown that the replacement of amide bonds
research in radiopharmaceutical and nuclear medicinal research
in the backbone of linear peptides by 1,2,3-triazole amide bond
for about the past two decades. As a result, the first radiolabeled
peptides (somatostatin analogues) have found routine
isosteres, readily available by the Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC, click chemistry),7,8 yields novel
applications in the clinic and are used in nuclear medicine as
peptidomimetics with promising properties for the development
of tumor-targeting radiotracers.9 We termed the systematic
diagnostic probes and radiotherapeutics for the management of
neuroendocrine tumors. In addition, a number of peptidic
replacement of amide bonds by 1,2,3-triazoles as trans-amide
radiotracers are currently in preclinical and clinical development.1
bond isosters a triazole scan. A triazole scan of a variant of the
agonistic BBN binding sequence, [Nle14]BBN(7–14),10 yielded
Bombesin (BBN) is an amphibian analog of the mammalian
gastrin-releasing peptide (GRP), a member of the family of
regulatory peptides, which target G-protein coupled receptors.
Specifically, BBN has a high affinity and specificity towards the
several novel peptidomimetics with retained nanomolar affinity
(KD) towards GRPR, an up to fivefold enhanced blood serum
stability in vitro and improved tumor targeting capabilities
GRP receptor (GRPR), a cell membrane receptor over-expressed
in vivo. In particular, amide-to-triazole substitution between the
amino acid residues Ala9Val10, Gly11His12 and the introduction
in a variety of clinically relevant tumors including prostate,
breast, colon, and small-cell lung carcinomas.2 As
a
of the heterocycle at the C-terminal Nle14 of [Nle14]BBN(7–14),
provided the most promising peptidomimetic BBN agonists
(Figure 1). In an effort to identify novel radiolabeled BBN
antagonists, we set out to apply our methodology to the
consequence, the 14 amino acid sequence of BBN (and
fragments thereof) has been widely studied for the development
of diagnostic and therapeutic radiopeptides for application in
nuclear oncology.3 Currently, two different classes of
radiolabeled BBN derivatives are under investigation. 1)
Agonistic versions that internalize into tumor cells by
Department of Radiology and Nuclear Medicine, University of Basel Hospital
endocytosis upon activation of the receptor and trigger an Division of Radiopharmaceutical Chemistry Petersgraben 4, 4031 Basel,
intracellular response (e.g., release of Ca2+). Historically,
Switzerland
radiolabeled GRPR agonists have been studied almost
exclusively based on the premise that radioactivity is
*Correspondence to: Thomas L. Mindt, Clinic of Radiology and Nuclear Medicine,
Division of Radiopharmaceutical Chemistry, University of Basel Hospital,
accumulated with higher efficiency in tumor cells (enhanced Petersgraben 4, 4031 Basel, Switzerland.
E-mail: Thomas.Mindt@usb.ch
tumor-to-background ratio) and that the proximity to the cell
† This article is published in the Journal of Labelled Compounds and
nucleus results in an improved efficacy where therapeutic
radionuclides are employed.4 2) Radiolabeled GRPR antagonists Radiopharmaceuticals as a special issue on ‘Current Developments in PET and
SPECT Imaging’, edited by Jonathan R. Dilworth, University of Oxford and Sofia
I. Pascu, University of Bath.
are not internalized into cells but exhibit a persistent attachment
to tumors resulting in an overall increased accumulation of
radioactivity at tumors and metastases in vivo.5 Even though
‡Additional supporting information may be found in the online version of this
the exact mode of action of the GRPR-targeting peptide article at the publisher’s web site.
J. Label Compd. Radiopharm 2014, 57 275–278
Copyright © 2013 John Wiley & Sons, Ltd.

I. E. Valverde et al.
Figure 1. Comparison of the structures of agonistic bombesin (BBN) derivative
[Nle14]BBN(7–14) and antagonist JMV594. Dashed lines illustrate the structural
similarity of the amino acid sequences, and arrows indicate positions where amide
bonds can be replaced by 1,2,3-triazoles in the agonist [Nle14]BBN(7–14) without
loss of the biological properties of the vector. Nle, norleucine; Sta, statine: (3S,4S)-
4-amino-3-hydroxy-6-methylheptanoic acid.
Scheme 1. Synthesis of α-azido acids; a) 3, K2CO3, CuSO4, MeOH, RT; b) LiOH,
MeOH/H2O (2:1), RT. For Val, R = CH(CH3)2; for His, R = CH2(1-trityl-1H-imidazole).
structurally closely related BBN receptor antagonist JMV594,5,11
a
peptide with high-sequence homology differing only in the last
two amino acids of the C-terminus and the additional DPhe at
the N-terminus. On the basis of our previous investigations with
[Nle14]BBN(7–14), we reasoned that a comparison with JMV594
would be most appropriate when the same linker, chelator, and
radionuclide are used. Thus, we employed a short, tetraethylene
glycol (15-amino-4,7,10,13-tetraoxapentadecanoic acid, PEG4-OH)
spacer for the N-terminal conjugation of the macrocyclic chelator
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA;
Table 1) and lutetium-177 (177Lu) as a therapeutic βÀ emitter with
a concomitant γ emission for imaging by single photon emission
computed tomography.
Boc-protecting groups of intermediates 12 and 14 with Fmoc
yielded α-amino alkynes 13 and 15 for solid phase synthesis.
Glycine analog 17 was prepared by Fmoc protection of
commercial propargyl amine (16).
The synthesis of reference compound 18 and 1,2,3-triazole
containing peptidomimetics 19–21 (Figure 2, Table 1) was
accomplished by standard Fmoc solid phase peptide synthesis
(Fmoc-SPPS) and CuAAC on solid support. In some cases,
fragments of the peptides were synthesized using an automated
synthesizer, whereas in other examples, amide couplings were
performed manually (Experimental). CuAAC on solid support was
performed according to published procedures employing Cu
[CH3CN]4PF6 as catalyst.14,15 After completion of the amino acid
sequence, the peptides were elongated N-terminally with a PEG4
spacer and functionalized with the universal macrocyclic chelator
DOTA. Following cleavage from the resin and deprotection, the
peptide conjugates 18–21 were obtained in high purity (>95%)
after preparative HPLC purification and structural identity was
verified by mass spectrometric analysis (Table 1).
Experimental
General methods, synthetic procedures, radiolabeling protocols, details
of in vitro experiments, and analytical data of intermediates and final
products can be found in the Supporting Information.
Results and discussion
Radiolabeling of DOTA conjugates 18–21 with 177LuCl3 was
performed using standard labeling procedures at 95°C in
NH4OAc (pH 5) as described by us and others.9,16 Radiolabeled
Building blocks required for the synthesis of 1,2,3-triazole-
containing peptidomimetics as indicated in Figure 1 were
prepared according to published procedures. Azide derivatives
of amino acids 4 and 6 were synthesized from the corresponding
amino acids 1 and 2 by a diazotransfer reaction employing the
reagent imidazole-1-sulfonyl azide (3; Scheme 1).12 In the case
of histidine (His) derivative 5, an additional saponification step
furnished desired α-azido acid 6. For the insertion of a mono-
substituted 1,2,3-triazole at the C-terminus of peptidomimetic
21 (Table 1), the azide functionality was introduced directly into
the linker of the resin on solid support as described in the
literature (Supporting Information).13
compounds
[
177Lu(18–21)] were obtained in excellent
radiochemical yield and purity (>95%) with a specific activity
of 0.05–0.3 mCi/nmol (not optimized).
With radiolabeled reference peptide
[
177Lu(18)] and
peptidomimetics [177Lu(19–21)] in hands, we next evaluated their
receptor binding and cell internalization properties as well as their
receptor affinity (KD) in vitro using GRPR overexpressing PC3 cells
(Figures 3 and 4). Receptor specificity was confirmed for all in vitro
studies described henceforth by blocking experiments in the
presence of an excess of natural BBN (Supporting Information).
Radiopeptide [177Lu(18)], used as a reference compound in this study,
α-Amino alkyne building blocks were prepared by multistep
synthesis involving the reduction of amino acid-derived Weinreb represents a novel antagonistic BBN radioconjugate. Despite
amides 8 and 10 with DIBAL-H followed by a Seyferth–Gilbert structural differences to published radiolabeled JMV594
homologation of the in situ generated α-amino aldehydes using derivatives,5,17
[
177Lu(18)] displayed receptor binding (approximately
the Bestmann–Ohira reagent 11 (Scheme 2). Exchange of the 30%) and cell internalization properties (approximately 8%) similar to
Table 1. Reference peptide 18 and 1,2,3-triazole containing peptidomimetics 19–21 obtained by Fmoc-SPPS and CuAAC on
solid support
Nr.
Structure
Yield (%)
[M + H]+ (calc.)
18
19
20
21
DOTA-PEG4-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2
DOTA-PEG4-DPhe-Gln-Trp-Alaψ[Tz]Val-Gly-His-Sta-Leu-NH2
DOTA-PEG4-DPhe-Gln-Trp-Ala-Val-Glyψ[Tz]His-Sta-Leu-NH2
DOTA-PEG4-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leuψ[Tz]-H
28
27
13
14
1746.932 (1746.943)
1770.957 (1770.954)
1770.944 (1770.954)
1770.993 (1770.954)
ψ[Tz] indicates the position of the amide-to-triazole substitution.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 275–278

I. E. Valverde et al.
Scheme 2. Synthesis of α-amino alkynes; a) MeONMe HCl, BOP, i-Pr2NEt, CH2Cl2, RT; b) i) DIBAL-H, CH2Cl2, À78°C; ii) dimethyl-1-diazo-2-oxopropylphosphonate, K2CO3,
MeOH, RT; c) trifluoroacetic acid/CH2Cl2 (2:1), RT; d) Fmoc-OSu, i-Pr2NEt, CH2Cl2, RT. For Ala, R = CH3; for Val, R = CH2CH(CH3)2; for Gly, R = H.
Figure 4. Receptor saturation assays of antagonistic 177Lu-labeled conjugates.
Data are presented as mean standard deviation (n = 2–3 in triplicates) and fitted
by nonlinear regression with GraphPad Prism 5.0.
that of reported derivatives. Interestingly, JMV594-derived [177Lu(18)]
exhibited to some extent cell internalization properties typically
Figure 2. Synthesis of 1,2,3-triazole containing bombesin (BBN) antagonistic
radiopeptidomimetics. SPPS: i) 20% piperidine/N,N,-Dimethylformadide (DMF); ii)
Fmoc-amino acid, azido acid, Fmoc-PEG4-OH, or DOTA(tBu)3, HATU, i-Pr2NEt; DMF;
a) For compound 21 (n = 0): 3, DMF; CuAAC: α-amino alkyne, [Cu(CH3CN)4]PF6,
observed for agonists even though the vector has been reported as
a full antagonist.11 Disappointingly, [177Lu(19–20)] did not bind to
i-Pr2NEt, DMF; b) trifluoroacetic acid/H2O/PhOH/i-Pr3SiH. SPPS, solid phase peptide
GRPR or internalize into PC3 cells, which indicates abolished receptor
affinity. On the other hand, compound [177Lu(21)] showed a similar
but less pronounced receptor binding (13%) and cell internalization
(3%) profile in comparison with reference compound [177Lu(18)].
Receptor saturation experiments revealed a receptor affinity for
synthesis; AA, amino acid.
[
177Lu(18)] and [177Lu(21)] of KD =2.7nMand KD =8.1nM, respectively.
The lower GRPR affinity of [177Lu(21)] compared with reference [177Lu
(18)] correlates with the observed decreased accumulation of the
1,2,3-triazole-modified compound [177Lu(21)] in PC3 cells.
The metabolic stability of [177Lu(18)] and [177Lu(21)] was
investigated by incubation of the compounds in human blood
serum at 37°C. Samples were taken within 1–48 h and analyzed
by γ HPLC after precipitation of proteins. Remarkably, both
antagonistic radiopeptides were still intact to a large extent (65%
and 75%, respectively) even after 48 h (Supporting Information).
This resistance to proteolytic degradation by serum proteases
is outstanding in comparison with radiolabeled BBN agonists,
which usually exhibit a serum half-life in the range of a few
hours.9,18 We hypothesize that the exceptional in vitro stability
of JMV594-derived antagonistic radioconjugates is at least in
part responsible for their observed favorable characteristics as
tumor-targeting radiopharmaceuticals.
Figure 3. Specific receptor binding and cell internalization of antagonistic 177Lu-
labeled peptide conjugates. Data are presented as mean standard deviation (n = 3
in triplicates) and fitted by nonlinear regression with GraphPad Prism 5.0.
J. Label Compd. Radiopharm 2014, 57 275–278
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

I. E. Valverde et al.
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Conclusions
In summary, we have applied the amide-to-triazole substitution
strategy of previously investigated agonistic GRPR-targeting
peptide [Nle14]BBN(7–14) to the structurally closely related
antagonistic peptide JMV594.
Despite the high-sequence homology of the two peptides
investigated, we observed that the majority of backbone
modifications tolerated by [Nle14]BBN(7–14) cannot be applied to
JMV594. Our results are in accordance with published data reporting
backbone modifications (N-methylation) of GRPR agonists and
antagonists based on the described amino acid sequences.19
The data obtained in this study provide an additional piece of
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triazole-containing GRPR-specific antagonistic radiopeptidomimetic.
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We thank Christiane A. Kluba, Alba Mascarin, Dr. Andreas
Bauman, and Sandra Vomstein (University of Basel Hospital) for
scientific discussions and technical assistance and the Cancer
League Basel and the Swiss National Science Foundation (grant
Nr. 132280 and 146385) for financial support.
Conflict of Interest
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The authors have no conflicts of interest.
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Supporting information
Additional supporting information may be found in the online
version of this article at the publisher’s web site.
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 275–278