Triazolo-peptidomimetics: novel radiolabeled minigastrin analogs for improved tumor targeting

Article abstract of DOI:10.1021/acs.jmedchem.9b01936

MG11 is a truncated analog of minigastrin, a peptide with high affinity and specificity toward the cholecystokinin-2 receptor (CCK2R), which is overexpressed by different tumors. Thus, radiolabeled MG11 derivatives have great potential for use in cancer d

Full text of DOI:10.1021/acs.jmedchem.9b01936

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Article
Triazolo-Peptidomimetics: Novel Radiolabeled
Minigastrin Analogs for Improved Tumor Targeting
Nathalie Grob, Daniel Häussinger, Xavier Deupi, Roger Schibli, Martin Behe, and Thomas L. Mindt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01936 • Publication Date (Web): 17 Apr 2020
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Triazolo-Peptidomimetics: Novel Radiolabeled
Minigastrin Analogs for Improved Tumor
Targeting
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Nathalie M. Grob,[a] Daniel Häussinger,[b] Xavier Deupi,[c,d]
Roger Schibli,[a,e] Martin Behe,*[e] and Thomas L. Mindt*[f,g,h]
a
Department of Chemistry and Applied Biosciences, ETH Zurich,
8093 Zürich (Switzerland)
b
Department of Chemistry, University of Basel, 4056 Basel
(Switzerland)
c
Condensed Matter Theory Group, Laboratory for Scientific
Computing and Modelling, Paul Scherrer Institute, 5232
Villigen (Switzerland)
d
Laboratory of Biomolecular Research, Division of Biology and
Chemistry, Paul Scherrer Institute, 5232 Villigen, Switzerland
e
Center for Radiopharmaceutical Sciences, Division of Biology
and Chemistry, Paul Scherrer Institute, 5232 Villigen
(Switzerland)
f
Ludwig Boltzmann Institute Applied Diagnostics, General
Hospital of Vienna, 1090 Vienna (Austria)
g
Department of Inorganic Chemistry, Faculty of Chemistry,
University of Vienna, 1090 Vienna (Austria)
h
Department of Biomedical Imaging and Image Guided Therapy,
Medical University of Vienna, 1090 Vienna (Austria)
KEYWORDS
Cancer, Peptidomimetics, Radiopharmaceuticals, Molecular
Modeling, Structure-Activity Relationships
ABSTRACT
MG11 is a truncated analog of minigastrin, a peptide with high
affinity and specificity towards the cholecystokinin-2 receptor
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(CCK2R), which is overexpressed by different tumors. Thus,
radiolabeled MG11 derivatives have great potential for use in
cancer diagnosis and therapy. A drawback of MG11 is its fast
degradation by proteases leading to moderate tumor uptake in
vivo. We introduced 1,4-disubstituted 1,2,3-triazoles as
metabolically stable bioisosteres to replace labile amide bonds
of the peptide. The “triazole scan” yielded peptidomimetics with
improved resistance to enzymatic degradation and/or enhanced
affinity towards the CCK2R. Remarkably, our lead compound
reached a 10-fold increase in receptor affinity resulting in a
2.6-fold improved tumor uptake in vivo. Modeling of the ligand-
CCK2R complex suggests that an additional cation-π interaction
of the aromatic triazole moiety with the Arg356 residue of the
receptor is accountable for these observations. We show for the
first time that the amide-to-triazole substitution strategy
offers new opportunities in drug development that go beyond the
metabolic stabilization of bioactive peptides.
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Introduction
Bioactive peptides are a valuable pool for drug discovery and
development, and the subject has been reviewed extensively.1, 2
Peptides are highly specific compounds, readily synthesized, and
the broad availability of structurally diverse building blocks
offers combinatorial options, which can be exploited to create
libraries of compounds for drug screening. A common drawback of
linear peptides for medical applications is their poor metabolic
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stability due to degradation by ubiquitous endo- and
exopeptidases. Different strategies for the enhancement of the
stability of peptides are known (e.g., cyclization, N-
methylation, or the use of D-, β-, or other non-canonical amino
acids), however often with variable outcomes.3 While many of
these modifications focus on the modification of the side chains
of amino acids, the use of bioisosteres for amide bonds, such
as triazoles, involve structural changes within the backbone of
the peptide. With their similar planarity and size, dipolar
moment, and comparable hydrogen-bonding features, 1,4- and 1,5-
disubstituted 1,2,3-triazoles have been identified as
metabolically stable bioisosteres of trans and cis amide bonds,
respectively.4-11 The systematic replacement of amide bonds in a
peptide sequence, one at a time, by a 1,4-disubstituted 1,2,3-
triazole has been termed a ‘triazole scan’. Valverde et al. and
Mascarin et al. demonstrated the suitability of this approach
by performing triazole scans of a bombesin (BBN) and a
neurotensin (NT) derivative, both targeting G-protein coupled
receptors (GPCRs) that are overexpressed by various tumors.12-14
The peptide conjugates were functionalized with the macrocyclic
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chelator
DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid) to enable labeling with radiometals.
Importantly, the synthesis of triazolo-peptidomimetics is
compatible with standard solid-phase peptide synthesis (SPPS).
Among the reported novel peptidomimetics of BBN and NT, several
candidates were identified with increased blood serum stability
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and preserved receptor affinity in vitro. The radiolabeled
compounds showed an increased uptake in tumors of mice bearing
xenografts, thus demonstrating the general utility of this
strategy.
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With these encouraging results in mind, we aimed to apply this
strategy to another peptide candidate targeting malignant
neoplasms such as, e.g., medullary thyroid cancer and small cell
lung cancer. The overexpression of the cholecystokinin-2
receptor (CCK2R) by various types of tumors was reported in the
late 1990s.15, 16 Ever since, several groups have investigated the
targeting of the CCK2R using analogs of cholecystokinin or
minigastrin labeled with different radionuclides.17-22
Eventually, the screening of ligands led to a multi-center
comparison of 12 different CCK2R-targeting peptide conjugates.23-
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The shortest peptide candidate included in these studies, MG11
(DOTA-DGlu10-Ala11-Tyr12-Gly13-Trp14-Met15-Asp16-Phe17-NH2), showed
promising receptor affinity but poor resistance against
metabolic degradation, resulting in an only moderate uptake in
tumors in vivo. We hypothesized that the poor stability24 of MG11
leads to substantial degradation of the radiotracer before the
target receptor at the tumor is reached. The short peptide
conjugate [Nle15]MG11 (Figure 1; Met15 of MG11 was replaced by
Nle15 to reduce the formation of oxidation side products during
radiolabeling)26 was chosen as the subject of a triazole scan
with full evaluation of the novel triazolo-minigastrins (TZMGs)
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in vitro (IC50, internalization, plasma stability, logDpH 7.4),
and in vivo (biodistribution with tumor-bearing mice).
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Figure 1. Structure of reference compound [Nle15]MG11 (top) with
numbering of the amide bonds. The colors of the numbers
correspond to those used in Figures 2-4 indicating the position
of the triazole, and structure of TZMG 6 bearing an amide-to-
triazole substitution at position 6 (bottom).
Results and Discussion
Synthesis and Radiolabeling with [177Lu]LuCl3 Starting from
protected amino acids and amino acid-derived alkyne building
blocks, all peptide conjugates were successfully synthesized on
solid support in yields of 10-74% and high purities (>95%) by
procedures adapted from Valverde et al. (Supporting Information
(SI), Table S4, pages S55-S71).12 Labeling of the DOTA-MG
derivatives with [177Lu]LuCl3 in ammonium acetate buffer at 95 °C
for 20 min provided the radiolabeled conjugates in radiochemical
yields and purities >95% (SI, pages S79-S82).
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The radionuclide Lu was chosen for this study due to its
concomitant emission of γ and β- radiation, which allows for
SPECT imaging and therapeutic applications. Depending on the
intended use (in vitro or in vivo experiments), molar activities
of 20-50 MBq/nmol were achieved (not optimized). The
radiolabeled conjugates were used for experiments without
further purification. As previously reported,12, 13 no significant
changes in logDpH 7.4 were observed resulting from to the amide-
to-triazole modification (Table 1).
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In vitro Evaluation The 177Lu-labeled compounds were incubated
in human blood plasma at 37 °C for 24 h to estimate their
resistance against enzymatic degradation. The plasma stability
experiment served as a model system, which is known to lack
membrane-bound proteases that contribute to the degradation of
peptides from the gastrin family.27-30 At different time points,
samples were taken and the metabolites were quantified by γ-
HPLC. Nonlinear regression (one-phase decay) was performed to
calculate the plasma half-life (t1/2, Table 1) of the compounds.
As already reported, reference compound [Nle15]MG11 showed fast
degradation in plasma with a t1/2 of 3.9 h (Figure 2).31 Prolonged
t1/2 were observed for six out of eight TZMGs showing that the
insertion of a triazole enhanced their resistance against
proteases. For TZMGs 1, 2, 3, 4, and 7, more than 50% compound
was found still intact after 24 h of incubation. The t1/2 of TZMG
4 was calculated to be 90 times longer than that of the reference
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compound indicating a substantial enhancement of its stability.
To our surprise, TZMGs 5 and 6 showed no benefit from the
modification in terms of stability. Three enzymatic cleavage
sites of unmodified MG11 are known (Tyr12-Gly13, Gly13-Trp14, and
Asp16-Phe17), and we assume that the placement of the triazole at
certain positions could enhance the access of enzymes to other
amide bonds.23, 32 This assumption is supported by the formation
of different metabolites depending on the position of the
triazole (SI, Figure S93).
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Figure 2: Percentage of intact 177Lu-labeled peptide conjugates
after incubation in human blood plasma for 24 h at 37 °C. Data
points show mean ± SD. n = 2-3, for values of t see Table 1.
½
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Figure 3: A) specific internalization of 177Lu-labeled reference
compound and TZMGs (n = 3-5 in triplicate) over 4 h. Data points
show mean ± SD. B) Comparison of membrane-bound and internalized
fraction after 30 min of incubation (box in Figure 3A),
representing total cell associated activity in relation to
applied radioactivity. Columns represent mean ± SD, *** for
p values ≤ 0.001.
For cell binding and internalization experiments, A431 cells
from human epidermoid carcinoma stably transfected to express
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the CCK2R (A431-CCK2R cells)33 were incubated with the Lu-
labeled conjugates for 4 h at 37 °C (Table 1). The cells were
washed, treated with acidic buffer and lysed by treatment with
NaOH after 30, 60, 120, and 240 min to determine free, membrane-
bound, and internalized fractions of the radiolabeled ligands
over time. An overview of the internalization of radiolabeled
TGMZs over 4 h and a comparison after 30 min are displayed in
Figure 3 A and B. Receptor specificity in vitro was demonstrated
for all compounds by blocking experiments in the presence of an
excess of minigastrin (see SI, Figure S95). The reference
compound showed a receptor-specific internalization of 32.2%
after 4 h of incubation. TZMGs 1-3 displayed no measurable or
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strongly reduced binding and internalization under the
experimental conditions. These three derivatives contain the
triazole modification within the four C-terminal amino acid
residues, which have been described as the minimal binding
sequence known to be very sensitive even for minor
modifications.34, 35 After 4 h, TZMGs 4 and 7 reached similar cell
internalization as the reference compound. Remarkably, TZMGs 5,
6, and 8 showed higher CCK2R-specific cell internalization after
4 h. TZMG 6 almost doubled its internalization compared to the
reference after 2 and 4 h. The effect was even more pronounced
at the earliest time point of 30 min (Figure 3B): While the
reference compound reached 11.7% of cell associated
radioactivity, TZMG 6 showed an exceptionally fast cell binding
and uptake of 31% of the applied radioactivity. The observed
faster internalization kinetics of TZMG 6 may be ascribed to
enhanced interactions with the CCK2R, or a transient, favorable
conformer of the peptide that allows for faster uptake.
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The affinity (IC50, Table 1) of the novel TZMGs towards CCK2R
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was determined by a competition-binding assay. Reported Lu-
labeled CCK2R-ligand PP-F11N (DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-
Asp-Phe-NH2)22 was dispensed to A431-CCK2R cells and challenged
by increasing concentrations of the TZMGs labeled with non-
nat
radioactive Lu3+ for 1 h at 4 °C. The cells were washed and
subsequently lysed to determine the bound fraction of [177Lu]Lu-
PP-F11N as a function of the concentration of added TZMG
competitors (for binding curves see the SI, Figure S94). The
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results mirror our observations from the cell internalization
experiments: In comparison to the reference compound [Nle15]MG11,
TZMG 1 showed a reduced receptor affinity by more than one order
of magnitude. TZMGs 2 and 3 could not displace in 50% of
[177Lu]Lu-PP-F11N even at the highest concentration tested (50
µM), which indicates an almost abolished affinity towards the
CCK2R. The binding curves of TZMGs 4, 5, and 7 closely resembled
the binding curve of the reference compound, resulting in
similar IC50 values. TZMG 8 had twice the affinity towards the
CCK2R than the reference compound, and TZMG 6 showed an almost
tenfold improvement of the IC50 value. The increased affinity of
TZMG 6 is in line with its superior cell internalization
characteristics. To the best of our knowledge, this is the first
report of a triazolo-peptidomimetic that exhibits a pronounced
increase in receptor affinity due to the subtle structural
modification.
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Table 1: Results from in vitro evaluation of TZMG derivatives.
SD = standard deviation, 95% CI = 95% confidence interval of
nonlinear regression; n= 3-5 in triplicates for internalization
and IC50, n= 2-3 for t1/2 and logDpH 7.4.
4 h specific
internalization
IC50 [mean,
(95% CI),
nM][b]
Plasma t1/2
[mean (95%
CI), h][a]
logDpH 7.4
[mean ±
SD][a]
Compound
[mean ± SD, %]
[a]
Reference,
15.4 (11.0 -3.9 (3.8 --3.86
21.1) 4.1) 0.18
±
±
±
32.2 ± 3.2
2.5 ± 2.9
0.1 ± 0.08
[Nle15]MG11[c]
200.5 (164 -34.9 (31.3 --3.69
TZMG 1
TZMG 2
245)
39.3)
0.28
35.9 (32.2 --3.95
40.3) 0.54
> 50 μM
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114.3 (96.5 --3.75
139.7) 0.40
±
±
±
±
±
±
±
±
TZMG 3
TZMG 4
TZMG 5
TZMG 6
TZMG 7
TZMG 8
βMG 9
0.2 ± 0.13
> 50 μM
25.4 (18.3 -349.8 (263 --3.98
34.6) 520) 0.43
33.1 ± 1.9
41.7 ± 3.9
54.3 ± 5.1
29.6 ±2.7
39.6 ± 2.7
44.5 ± 3.3
37.4 ± 3.7
15.6 (12.3 -3.8 (3.6 --4.00
19.7) 4.0) 0.35
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1.7 (1.3 -2.6 (2.5 --4.06
2.3) 2.7) 0.11
20.9 (17.0 -51.4 (46.2 --3.90
25.7) 57.7) 0.18
8.0 (6.3 -7.7 (7.3 --3.98
10.9) 8.1) 0.45
18.8 (15.3 -8.5 (8.0 --3.73
22.8) 9.0) 0.24
25.8 (20.9 -4.7 (4.4 --3.83
βMG 10
31.4)
5.1)
0.21
[a]
[b]
Results obtained with [177Lu]Lu-labeled compounds.
Results obtained
with non-radioactive natLu-labeled compounds.
Data of [Nle15]MG11were
[c]
previously published and are included for a comparison.31
In vivo Evaluation The biodistribution of TZMGs 4-8 with
retained receptor affinity and cell internalization properties
in vitro was evaluated in mice with CCK2R-positive tumor
xenografts and compared with the reference compound [Nle15]MG11.
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4 h post injection (p.i.) of the Lu-labeled compounds, the
animals were sacrificed, dissected, the organs of interest were
weighed, and the radioactivity was determined with a γ-counter.
Excess amounts of minigastrin were co-injected to block
receptor-mediated uptake in four out of eight animals per
compound, demonstrating CCK2R-specificity in vivo. All compounds
showed nearly complete clearance from the blood within 4 h
(Figure 4; for tables including data of all organs and results
of blocking experiments see SI, Tables S12-18). Liver and
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intestines did not retain the radiotracers in significant
amounts. The uptake in the kidneys was not blocked by co-
injection of minigastrin, indicating that the accumulation of
radioactivity is not mediated by the CCK2R but is due to renal
excretion.36 On the other hand, co-injection of minigastrin
reduced significantly the accumulation of radioactivity in
CCK2R-positive stomach and tumor, which confirms receptor-
mediated uptake of the radiotracer. All other organs showed
negligible background uptake both with and without co-injection
of the blocking agent.
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Figure 4: Biodistribution of 177Lu-labeled reference compound and
TZMGs 4-8 in mice 4 h p.i. % of injected activity per gram of
organ/tissue (% i.A./g) is shown for blood, liver, intestines,
kidneys, stomach, and tumor without (plain columns) and with co-
injection of excess minigastrin (crosshatched columns). n = 4
animals per group * for p values < 0.033, *** for p values <
0.001. For complete tables of biodistribution data for all
collected organs and tissues see SI, Tables S12-S18.
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Focusing on the tumor uptake, the superiority of TZMG 6 becomes
evident, as replacement of the amide bond between Tyr12 and Gly13
by a 1,4-disubstituted 1,2,3-triazole resulted in a 2.6-fold
increased uptake in the tumor xenografts (3.91 ± 1.48 % i.A./g
for TZMG 6 vs 1.48 ± 0.73 % i.A./g for the reference compound).
While stabilized TZMGs 4, 5, 7, and 8 showed similar tumor uptake
as the reference compound, TZMG 6 reached the highest value, in
spite of its reduced plasma stability in vitro (Table 1). The
increased tumor-to-kidney ratio of TZMG 6 (5.81 ± 2.80 versus
1.83 ± 0.88 for the reference compound) is beneficial for
translation of the compound to the clinic, as the radiation dose
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to the kidney – and thus potential nephrotoxicity – is of concern
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for PRRT.37,
TZMG 6 exhibited an increased rate of cell
internalization in vitro; if this also holds true in vivo, it
could explain the high tumor uptake observed in vivo. As peptidic
radiotracers are rapidly eliminated from the blood by glomerular
filtration, an improved and accelerated receptor interaction may
constitute an advantage.
Biodistributions at additional time points (1 and 24 h p.i.)
were performed with the 177Lu-labeled reference compound
[Nle15]MG11 and TZMG 6, as this derivative performed best in the
preceding studies. Figure 5 highlights the uptake of the
radiotracers in CCK2R-positive stomach and tumor, and the
kidneys (for tables including data of all organs and tumor-to-
organ/tissue ratios see SI, Tables S19-S20). For both compounds,
the highest tumor uptake was observed after 1 h, which rapidly
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decreased for the reference compound over time (<0.9 % i.A./g
after 24 h). At all time points, TZMG 6 displayed a significantly
higher uptake of radioactivity in the tumor, which was in a
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similar range as compared to recent reports of other stabilized
40
minigastrin derivatives.39,
We also observed a slower tumor
washout for TZMG 6, which is an important feature for potential
therapeutic applications. In the direct comparison, only 34% of
the initial activity in the tumor remained for the reference
compound, whereas 75% of the tumor activity was still present
for TZMG 6 24 h after injection. The in vivo results from these
extended studies underline the improved tumor-targeting
characteristics of TZMG 6 and thus, its potential for clinical
translation.
Figure 5: Biodistribution of the reference compound and TZMG 6
at 1, 4, and 24 h p.i., n = 4 animals per group, * for p values
< 0.033, *** for p values < 0.001. For complete tables of
biodistribution data and tumor-to-tissue/organ ratios see SI,
Tables S19-S20.
Structural analyses Intrigued by the unique features of TZMG
6, we set out to study the effect of the amide-to-triazole
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substitution in the position Tyr12-Gly13 of [Nle15]MG11 in more
detail. Even though 1,4-disubstituted 1,2,3-triazoles are well-
suited bioisosteres of trans-amide bonds, small structural and
physico-chemical differences are to be expected. For instance,
replacement of an amide bond by the heterocycle increases the
distance between the side chains of adjacent amino acids by
approx. 1.1 Å.11 In addition, the triazole has a higher dipolar
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moment (5 D vs 3.7 D for amide bonds) and can be engaged in π-
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interactions.41,
To investigate the effect of the structural
modification in TZMG 6, we prepared the analogs βMG 9 and βMG
10 in which Gly13 and Tyr12 were replaced by Ala13 and homoTyr12,
respectively. Insertion of the -amino acids results in a
similar distance between the side chains of the respective amino
acids as in the case of TZMG 6 (SI, Figure S98).43 Because the
in vitro characteristics of βMG 9 and βMG 10 were similar to
those of the reference compound [Nle15]MG11 (Table 1; SI Figure
S97 A-C), an increase in the distance between the side chains
of Tyr12-Gly13 could be excluded as a reason for the observed
superior properties of TZMG 6.
Next, we studied the effect of the triazole modification on
the conformation of the peptide in solution by circular
dichroism (CD) and NMR experiments. CD spectra of TZMG 6 and the
reference compound indicated that the peptides do not adopt a
defined structure in solution but have a random coil
conformation (SI, Figure S96), which is in agreement with
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published data of related minigastrin derivatives.39,
The
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conformations were further investigated by NMR: We assigned all
1H and 13C chemical shifts of TZMG 6 and [Nle15]MG11, both with
the empty DOTA chelator, as well as complexed with non-
radioactive natLu3+ (see Tables S10-11 and Figures S77-S80). The
assignment was carried out with routine COSY, ROESY, HSQC, and
HMBC spectra, and the ROE intensities were converted into
distance restraints. Structure calculations using these
restraints were performed with the XPLOR-NIH software package45,
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and confirmed random coil conformations in all cases.39, 47
Figure 6: A) Three-dimensional structural homology model of the
CCK2R (shown as ribbons) bound to TZMG 6 (spheres) in a lipid
bilayer (yellow). (B) Detail of TZMG 6 in the receptor binding
pocket. The C-terminal residues Trp14-Nle15-Asp16-Phe17 are
colored in magenta, the N-terminal residues DGlu10-Ala11-Tyr12 are
colored in green, and the connecting 1,2,3-triazole moiety is
colored in yellow. The side chain of residue Arg356 is shown as
sticks situated in close proximity (5.0-5.5 Å) to Tyr12-Ψ[Tz]-
Gly13. The previously reported CCK2R homology model is based on
39
PDB entries 4ZJ8 and 4S0V (see also SI).
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Despite great recent advances in structural determination of
GPCRs,48 the three-dimensional structure of the CCK2R and the
precise binding mode of cholecystokinin, gastrin, and related
peptides to this receptor are not yet known. However, several
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mutagenesis and modeling studies have identified essential
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regions and residues for ligand binding.39,
In order to
get a better understanding of the properties of TZMG 6, we built
a computational model of this ligand bound to the CCK2R (Figure
6A and SI, page S90).39 The C-terminal part of the peptide (Trp14-
Nle15-Asp16-Phe17-NH2) forms a short turn buried in the
orthosteric binding site (Figure 6B), where it can interact
with, e.g., residues Arg57, His376, His207, and Arg356. The N-
terminal part (DGlu10-Ala11-Tyr12) extends towards the
extracellular domains of the receptor, where it can interact
with residues in a ‘vestibule’ formed by, e.g., residues Arg45,
Arg50, Arg201 and Arg365.39 In gastrin-derived peptides, these two
elements are joint by Gly13. Remarkably, the triazole ring of
TZMG 6 at this position (Tyr12-Ψ[Tz]-Gly13) is able to retain the
local structure without major distortions of the backbone (SI,
Figure S99).
According to our model, Arg356 is part of the TZMG 6 binding
pocket (Figure 6B). We hypothesize that the charged guanidino
moiety of the arginine side chain forms a cation-π interaction
with the aromatic triazole heterocycle. The distance between the
two moieties was measured to be 5.0-5.5 Å, well within the
proximity requirements for such interactions (<6.0 Å).53 Cation-π
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interactions can significantly contribute to the binding
affinity of the ligand and could account for the remarkable
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properties of TZMG 6 observed in vitro and in vivo.54,
9
Interestingly, Arg356 has been suggested to be involved in a
cation-π interaction with the aromatic ring of Tyr12 of a related
minigastrin analog.43
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Amongst other interesting properties, 1,2,3-triazoles are also
hydrogen bond donors and acceptors.56 However, in our model of
TZMG 6 bound to the CCK2R, the triazole between the C-terminal
turn and the N-terminal part of the ligand has no clear intra-
or inter-molecular partners to fulfill its hydrogen- bonding
potential. The lack of improvement in binding affinity of the
-amino acid analogs MG 9 and MG 10 also suggests that hydrogen
bond interactions with the backbone of TZMG 6 is not a major
contributor to its improved receptor affinity and cellular
uptake.
In summary, we hypothesize that the additional cation-π
interaction enabled by the 1,2,3-triazole moiety is the reason
for the observed improved tumor-targeting properties of TZMG 6.
The aromaticity of the triazole amide-bond bioisostere and its
potential for π–bonding interactions are rarely discussed in
this context. Based on our experimental data, we conclude that
the additional cation-π interaction is likely the main factor
that provides an explanation to our experimental observations
of TZMG 6 in vitro and in vivo. The structural analysis and
computational modeling provide new valuable insights into the
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possible molecular mode of action of our lead compound TZMG 6,
and we expect that the knowledge gained in this study will serve
as the base for future developments of new radiotracers
targeting the CCK2R.
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Conclusion
We successfully performed a triazole scan with [Nle15]MG11 to
create eight minigastrin triazolo-peptidomimetics. Some of these
novel analogs of radiolabeled minigastrin showed improved
biological properties, either in pharmacodynamic parameters such
as receptor affinity and cell internalization, or in their
pharmacokinetic behavior regarding resistance to metabolic
degradation. To the best of our knowledge, we present here for
the first time a peptidic radioconjugate in which the triazole
modification has a decisively positive impact on the receptor
affinity of a peptide conjugate, which in turn results in
superior tumor targeting in vivo. Unlike in previous studies
with triazole analogs of bombesin and neurotensin, the metabolic
stability of the peptidomimetic conjugates played a secondary
role in vivo in the case of [Nle15]MG. Structural elucidation by
CD, NMR, and computational modeling support our hypothesis of
an altered binding mode of TZMG 6 with an additional cation-π
interaction between the Arg356 residue of the CCK2R binding
pocket and the aromatic triazole moiety placed in the backbone
of the ligand. The work described herein highlights the general
utility of the amide-to-triazole substitution for the
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development of bioactive peptide analogs with improved tumor-
targeting properties.
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Experimental Section
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Synthesized building blocks (α-amino alkynes) were analyzed by
ESI-HRMS, 1D and 2D NMR (1H, 13C, COSY, HSQC, HMBC). Enantiomeric
purities of building blocks were determined by either chiral
HPLC or formation of diastereomers followed by NMR analysis.
Peptides (mono-TZMGs) were analyzed by RP-HPLC and ESI-HRMS and,
after radiolabeling with [177Lu]Lu3+, by -HPLC. The purity of all
final compounds including those used for in vitro and in vivo
experiments is >95%.
Materials and Methods. Reagents were purchased from Sigma
Aldrich (Buchs, Switzerland), Thermo Fisher Scientific (Zürich,
Switzerland), Acros Chemicals (Geel, Belgium), Tokyo Chemical
Industries (Eschborn, Germany), Fluka (Buchs, Switzerland), ABCR
Chemicals (Karlsruhe, Germany), Merck (Darmstadt, Germany),
AppliChem (Darmstadt, Germany), BioConcept (Allschwil,
Switzerland), VWR or Avantor Performance (Dietikon, Switzerland)
and used without further purification. Fmoc-protected amino
acids and Rink amide MBHA LL resin (0.30–0.40 mmol/g, 100-200
mesh) were bought from Bachem (Bubendorf, Switzerland) or
NovaBiotech (Darmstadt, Germany). Fmoc-βhomoTyr(tBu)-OH was
ordered from Fluorochem (Derbyshire, UK). DOTA-tris(tBu)ester
was purchased from CheMatech (Dijon, France). Fmoc-Trp(Boc)-CCH
has previously been synthesized by Dr. Ibai Valverde and was
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kindly provided. [177Lu]LuCl3 was purchased from ITG (Garching,
Germany). Human blood plasma (A+) was ordered from the cantonal
blood donation center (Aarau, Switzerland). Minigastrin (H-
LEEEEEAYGWMDF-NH2) for blocking experiments and CCK2R ligand PP-
F11N (DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2)22, were
purchased from Peptide Specialty Laboratories GmbH (Heidelberg,
Germany).
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UV-measurements for Fmoc/tBu solid-phase peptide synthesis
(SPPS) were conducted on an Agilent Technologies Cary 60 UV-Vis
spectrophotometer. The scale and yield of the SPPS were
determined by quantification of the fluorenylmethylpiperidine
adduct at λ = 301 nm (ε = 7800 L∙mol-1∙cm-1). Measurements of UV
absorption at λ = 280 nm on an IMPLEN NanoPhotometer P360 were
used to determine the concentrations of peptide conjugate stock
solutions (ε = 6990 L∙mol-1∙cm-1; calculated for H-EAYGWMDF-NH2
by ExPASy – ProtParam of the Swiss Institute of Bioinformatics).
HPLC was performed on a Merck-Hitachi LaChrom D-7000 HPLC-system
monitored by UV-detection at 215 nm. γ-HPLC was performed on the
same system equipped with a Berthold HPLC Radioactivity monitor
LB 506 C-1 or on a Varian ProStar HPLC system monitored by UV-
detection at 215 nm and Radiomatic Flo-One Beta radio detector
from Packard. A ReproSil Chiral-NR column (PN:r18.nr, 8 μm, 250
x 4.6 mm (analytical) or 10 mm (semipreparative), Dr. Maisch
GmbH, Ammerbuch-Entringen, Germany) was used for chiral HPLC
using isopropanol and hexane as mobile phases A and B,
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respectively. A Jupiter Proteo column (90 Å, 4 μm, 250 x 4.6 mm
(analytical) or 10 mm (semipreparative), Phenomenex,
Aschaffenburg, Germany) was used for peptide analytics and
peptide purification with CH3CN (0.1% TFA) and H2O (0.1% TFA) as
mobile phases A and B, respectively. Quality control of
radiolabeling was performed with a ReproSil-Pur C18-AQ column
(3 μm, 100 x 4.6 mm, Dr. Maisch, Ammerbuch-Entringen, Germany),
and a Supelco Discovery C18 column (5 μm, 250 x 4.6 mm, Sigma-
Aldrich, St. Louis, MO, USA) was used for plasma stability
studies with CH3CN (0.1% TFA) and H2O (0.1% TFA) as mobile phases
A and B, respectively.
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High resolution mass spectrometry (HRMS) measurements were
recorded on a Bruker maXis (UHR-TOF) with ESI ion source and Qq-
TOF analyzer in positive mode and processed by the software
Compass 1.5, HyStar 3.2.44, DataAnalysis 4.1 and BioTools 3.3.
Low resolution mass spectrometry (LRMS) was measured on a Waters
Acquity UPLC H-class system over an Acquity UPLC Peptide BEH C18
column (300 Å, 1.7 μm, 2.1 mm x 50 mm) with a Xevo G2-SX QTOF
detector and evaluated by Mass Lynx software version 4.1.
Optical rotations were measured at 20 °C with an Anton Paar MCP
300 polarimeter in the specified solvent. Circular dichroism
(CD) spectra were recorded at rt between 190 and 320 nm on a
Chirascan Plus Circular Dichroism Spectrometer by Applied
Photophysics Ltd.
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Nuclear magnetic resonance (NMR) spectra of α -amino alkynes,
Weinreb amides and α-amino alcohols were recorded on a Bruker
400 MHz spectrometer at rt (298 K). Chemical shifts are reported
in parts per million (ppm) with the corresponding solvent
signals as internal reference. Values of the coupling constant,
J, are given in hertz (Hz); the following abbreviations are used
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in this section for the description of the H-NMR spectra:
singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), doublet of doublet (dd), triplet of doublet (td) and bs
(broad signal). Tert. C, quart. C, and aromat. C(H) are used as
abbreviations for tertiary, quaternary, and aromatic carbons,
13
respectively, for the description of the C-NMR spectra. The
chemical shifts of complex multiplets are given as the range of
their occurrence. Further assignments were achieved by using 2D
NMR experiments when appropriate (cosy, hsqc, hmbc). Residual
solvent signals of EtOAc, DCM, and water were considered in the
yield calculations.
(Radio-)Metal-labeling was conducted in 1.5 mL protein low-
binding Eppendorf tubes. An Eppendorf Thermomixer comfort was
used for heating and simultaneous shaking of the labeling
mixture. An Eppendorf Centrifuge 5415 C was used for
centrifugation during in vitro experiments.
Quantitative -counting was performed by a Packard COBRA-II
Auto-Gamma counter by measuring energy windows A (50-400 keV)
and B (190-230 keV) for 1 min.
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The A431-CCK2R cells were kindly provided by Dr. Aloj
(Department of Radiology, University of Cambridge, Cambridge,
United Kingdom).
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Female CD1 nu/nu mice were bought from Charles River
Laboratories (Sulzfeld, Germany). All animal experiments were
performed in compliance with Swiss laws on animal protection and
approved by the cantonal Veterinary Office of the canton of
Aargau (license AG 75700).
NMR experiments for structure determination of TZMG 6 and
[Nle15]MG11 were performed on a Bruker Avance III NMR
spectrometer operating at 600.13 MHz proton frequency. The
instrument was equipped with a cryogenic 5-mm QCI indirect
quadruple resonance probe (H/C/N/F/D) or with a direct observe
5-mm BBFO smart probe, both using shielded z-gradient coils and
a GAB gradient amplifier (10 Ampere, maximum gradient strength
52.5 G/cm). The experiments were performed at 298 K, and the
temperature was calibrated using a methanol standard showing
accuracy within +/- 0.2 K. Standard pulse sequences were used
for the cosy, tocsy, roesy, hsqc, and hmbc two-dimensional NMR
experiments. Watergate water suppression methods were applied
for the samples in H2O.
Both constructs were assigned with and without Lu3+, in H2O and
in D2O as solvents and 10 mM phosphate buffer at a pH / pD of
7.40. ROE correlations were converted into distance restraints,
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and the structures were determined by a standard simulated
annealing protocol using the software package Xplor-NIH.45, 46
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Synthesis. Protected α-amino alkynes Fmoc-Phe-CCH, Fmoc-Nle-
CCH, Fmoc-Trp(Boc)-CCH, Fmoc-Gly-CCH, Fmoc-Tyr(tBu)-CCH, and
Fmoc-Ala-CCH were synthesized following previously published
methods.12, 13 Fmoc-Asp(OtBu)-CCH (Alkyne 9) and Fmoc-DGlu(OtBu)
(Alkyne 16) were synthesized in two steps as described in the
Supporting Information.
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Manual synthesis of the peptide conjugates was performed as
previously reported.12, 13 In brief, Fmoc-amino acids were coupled
on a Rink Amide MBHA LL according to General Procedure A. A
diazotransfer following General Procedure B on the deprotected
N-terminal amine yielded the azide precursor for the CuAAC. The
1,4-disubstituted 1,2,3-triazole was obtained using General
Procedure C. The sequence was elongated by coupling of the
remaining amino acids and finished by conjugation of DOTA-
tris(tBu) using General Procedure A. Resin cleavage and
deprotection was achieved following General Procedure D.
General Procedure A, manual solid-phase peptide synthesis: Rink
amide MBHA low loading resin (ca. 100 mg, 0.03 – 0.04 mmol) was
placed in a polypropylene syringe with a polyethylene frit
fitted with a Teflon tap and swollen repeatedly in CH2Cl2 and
DMF. 20% piperidine in DMF was used to cleave the Fmoc protecting
group (3 x 3 min, rt). For elongation of the sequence, Fmoc-
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protected amino acids or DOTA-tris(tert-butyl ester) (2 equiv.,
0.06 mmol), HATU (1.9 equiv., 0.057 mmol), and DIPEA (5 equiv.,
0.15 mmol) in DMF (total 3 mL) were added to the resin. The
suspension was shaken for 1 h at rt. The solvent was removed by
filtration, and the resin was repeatedly washed with DMF and
CH2Cl2. Completion of the reaction was monitored by the Kaiser
test57, and coupling was repeated if necessary.
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General Procedure B, introduction of the azide functionality on
solid support: After cleavage of the Fmoc protecting group, the
free N-terminal amine was treated with imidazole-1-sulfonyl
azide hydrochloride (3 equiv., 0.09 mmol), DIPEA (9 equiv., 0.27
mmol), and a catalytic amount of CuSO4 (0.01 equiv., 0.03 µmol)58
in DMF (total 2 mL). The suspension was shaken for 1 h at rt.
The solvent was removed by filtration, and the resin was
repeatedly washed with a solution of 0.5% sodium
diethyldithiocarbamate in DMF, DMF, and CH2Cl2. Completion of
the reaction was monitored by the Kaiser test57, and the reaction
was repeated if necessary.
CAUTION: Occasional instability of imidazole-1-sulfonyl
azide∙HCl has been reported and safer alternatives were
developed since.59
General Procedure C, CuAAC on solid support: The Fmoc-protected
α-amino alkyne (2 equiv., 0.06 mmol), DIPEA (1 equiv., 0.03
mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.5
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equiv., 0.015 mmol), and TBTA (0.5 equiv., 0.015 mmol) in DMF
(2 mL) were added to the N-terminal azide functionality on solid
phase, and the suspension was shaken for 12-14 h at rt. The
solvent was removed by filtration, and the resin was repeatedly
washed with a solution of 0.5% sodium diethyldithiocarbamate in
DMF, DMF, and CH2Cl2. Completion of the reaction was monitored
by a colorimetric test for aliphatic azides60.
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General Procedure D, cleavage from the resin and deprotection:
After the final coupling of the N-terminal chelator DOTA-
tris(tert-butyl ester), the conjugates were deprotected and
cleaved from the resin using TFA/TIS/H2O/phenol
(92.5/2.5/2.5/2.5, 6 mL) with agitation for 5 h at rt. The
cleavage mixture was separated from the resin by filtration, and
a stream of nitrogen was applied for evaporation of the volatile
components. The crude peptide was then precipitated by addition
of ice-cold diethyl ether (15 mL). After centrifugation (1800
rpm, 540 RCF, 5 min) and two washing steps with ice-cold diethyl
ether, the crude peptide conjugates were dissolved in 20% CH3CN
in water (1 mg/mL) and purified by reverse phase semipreparative
HPLC. Subsequent lyophilization gave the final products as
fluffy, white solids.
[Nle15]MG11: The synthesis of the reference compound has been
previously described by the authors.31 The reference peptide
conjugate bearing no triazole modification was prepared
following General Procedures A and D using Rink amide MBHA low
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loading resin (0.057 mmol) and commercially available Fmoc-Phe-
OH, Fmoc-Asp(OtBu)-OH, Fmoc-Nle-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-
OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-DGlu(OtBu)-OH, and DOTA-
(tris-tBu). [Nle15]MG11 was obtained as a white solid in high
purity (>99%) after purification by semipreparative HPLC (30-
40% A in B in 15 min) and subsequent lyophilization (15 mg,
71%). ESI-HRMS m/z [M+H+]+ calculated for C65H89N14O20: 1385.6372,
found: 1385.6370.
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TZMG 1: Peptide conjugate TZMG 1 was prepared following General
Procedures A-D using a Rink amide MBHA low loading resin (0.051
mmol) and commercially available Fmoc-Asp(OtBu)-OH, Fmoc-Nle-
OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-
OH, Fmoc-DGlu(OtBu)-OH, and DOTA-(tris-tBu). Fmoc-Phe-CCH
(Alkyne 8) was used as a triazole precursor for the CuAAC.
Peptide conjugate TZMG 1 was obtained as a white solid in high
purity (>99%) after purification by semipreparative HPLC (25-
35% A in B in 15 min) and subsequent lyophilization (6 mg, 35%).
ESI-HRMS m/z [M+H+]+ calculated for C66H88N16O19: 1409.6484, found
1409.6506.
TZMG 2: Peptide conjugate TZMG 2 was prepared following General
Procedures A-D using a Rink amide MBHA low loading resin (0.0379
mmol) and commercially available Fmoc-Phe-OH, Fmoc-Nle-OH, Fmoc-
Trp(Boc)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-
DGlu(OtBu)-OH, and DOTA-(tris-tBu). Fmoc-Asp(OtBu)-CCH (Alkyne
9) was used as triazole precursor for the CuAAC. Peptide
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conjugate TZMG 2 was obtained as a white solid in high purity
(98.5%) after purification by semipreparative HPLC (20-30% A
over 30 min) and subsequent lyophilization (1.7 mg, 10%). ESI-
HRMS m/z [M+H+]+ calculated for C66H88N16O19: 1409.6484, found
1409.6463.
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TZMG 3: Peptide conjugate TZMG 3 was prepared following General
Procedures A-D using a Rink amide MBHA low loading resin (0.040
mmol) and commercially available Fmoc-Phe-OH, Fmoc-Asp(OtBu)-
OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-
OH, Fmoc-DGlu(OtBu)-OH, and DOTA-(tris-tBu). Fmoc-Nle-CCH
(Alkyne 11) was used as triazole precursor. Peptide conjugate
TZMG 3 was obtained as a white solid in high purity (> 99%)
after purification by semipreparative HPLC (25-35% A in B in 15
min, 4 ml/min) and subsequent lyophilization (6 mg, 76%). ESI-
HRMS m/z [M+2H+]2+ calculated for C66H88N16O19: 705.3279, found
705.3287.
TZMG 4: Peptide conjugate TZMG 4 was prepared following General
Procedures A-D using a Rink amide MBHA low loading resin (0.041
mmol) and commercially available Fmoc-Phe-OH, Fmoc-Asp(OtBu)-
OH, Fmoc-Nle, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-
DGlu(OtBu)-OH, and DOTA-(tris-tBu). Fmoc-Trp(Boc)-CCH was used
as triazole precursor. Peptide conjugate TZMG 4 was obtained as
a white solid in high purity (> 99%) after purification by
semipreparative HPLC (25-35% A in B in 15 min) and subsequent
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