A fluorescent probe for investigating the activation of anticancer platinum(IV) prodrugs based on the cisplatin scaffold

Article abstract of DOI:10.1002/anie.201305734

Watching closely: A fluorescent probe was engineered to detect the clinically relevant platinum drug cisplatin within a complex cellular environment, thus providing a direct means for visualizing its cell entry and the activation of platinum(IV) prodrugs in cancer cells. Copyright

Full text of DOI:10.1002/anie.201305734

Angewandte
Chemie
DOI: 10.1002/anie.201305734
Cellular Imaging
A Fluorescent Probe for Investigating the Activation of Anticancer
Platinum(IV) Prodrugs Based on the Cisplatin Scaffold**
Diego Montagner, Siew Qi Yap, and Wee Han Ang*
In memory of Elena Bertacco
Following the discovery of its potent antitumoral activity in
965, the inorganic drug cisplatin has become one of the most
accumulation of these clinically important compounds are
lacking. Herein, we describe a fluorescent probe that was
custom-built for the detection of Pt drugs such as cisplatin.
1
[1]
II
important anticancer agents in clinical use. It exhibits
exceptional activity against germ-cell testicular cancer, for
Using confocal microscopy, we could determine the local-
[2]
IV
which it is the first-line therapeutic option. Its success led to
the development of second-generation drugs, namely carbo-
platin and oxaliplatin, both of which are also based on the cis-
diam(m)ineplatinum(II) pharmacophore. Together, these
ization of the Pt prodrugs in cancer cells in vitro after cell
entry and intracellular reduction.
IV
Elucidating how Pt prodrugs are processed after cell
entry would significantly improve our understanding of these
compounds. Hambley et al. used elemental tomography to
II
FDA-approved Pt drugs constitute some of the most
[3]
IV
widely used chemotherapeutic agents (Figure 1).
Yet,
map the uptake of Pt prodrug complexes by tumor
spheroids. Using X-ray near edge adsorption spectroscopy
IV
(
XANES), they showed that cancer cells treated with Pt
IV
II
[10]
prodrugs contained both Pt and Pt species. However,
these techniques required synchrotron X-ray radiation, and so
cannot be applied to living cells. Elemental-analysis tech-
niques, such as graphite-furnace atomic-absorption spectrom-
etry (GF-AAS) and inductively coupled plasma mass spec-
trometry (ICP-MS), have also been used extensively to
quantitate the Pt distribution in tissue or cellular compart-
II
IV
Figure 1. FDA-approved Pt drugs and a Pt prodrug evaluated in
clinical trials.
[
11]
ments, but they cannot be applied in cellular imaging. More
recently, NanoSIMS was used to map the accumulation of
cisplatin and the multinuclear Pt drug candidate TriplatinNC
in vitro, but it cannot distinguish between different Pt
oxidation states (NanoSIMS = nanoscale secondary ion
II
despite their clinical success, the use of Pt drugs is limited
by their high toxicity, severe side-effects, and incidences of
[
4]
drug resistance. To address some of these limitations,
researchers have developed stable Pt carboxylate com-
IV
[12]
mass spectrometry).
plexes as anticancer prodrugs that can be activated by
intracellular reduction to release their latent cytotoxic
activity. This versatile strategy has been used to develop
Fluorescence microscopy remains the most effective and
accessible method for imaging at the cellular level. Using
immunofluorescence, Lippard and co-workers demonstrated
[
5]
IV
[6]
IV
Pt prodrug complexes with highly tuned properties, which
that, after administration, Pt prodrugs were capable of
[7]
are capable of targeting, as well as delivering novel modes of
action. The most prominent example is satraplatin, which is
currently undergoing Phase III clinical trials against hor-
mone-refractory prostate cancer (Figure 1). Although there
is much interest in understanding and exploiting this prodrug
strategy as a springboard towards the next generation of
Pt drugs, tools capable of directly visualizing the uptake and
forming nuclear Pt–DNA adducts in vitro, which were their
[8]
[13]
biological target.
To visualize the reduction process,
IV
fluorophores have also been attached directly to the Pt
scaffold, resulting in fluorescence quenching of its Pt
[
9]
II
[14]
congener. Reduction was accompanied by a concomitant
enhancement in fluorescence intensity, which, in principle,
provides the most direct method for visualizing reduction. Pt
complexes have also been labeled, either directly or through
the use of post-labeling strategies, with fluorophores to study
[
+]
[+]
[
*] Dr. D. Montagner, S. Q. Yap, Prof. Dr. W. H. Ang
Department of Chemistry, National University of Singapore
[15]
their intracellular distribution. However, modification of
IV
the Pt complexes with such bulky organic groups changes
3
Science Drive 3, Singapore (Singapore)
the pharmacophore and alters their uptake characteristics,
thus rendering them different from cisplatin.
E-mail: chmawh@nus.edu.sg
+
[
] These authors contributed equally to this work.
Therefore, an ideal imaging probe should be delinked
from the Pt scaffold and yet be selective enough to distinguish
[
**] We acknowledge generous funding provided by A*STAR (TSRP-
iNPBi 1021520016), the National University of Singapore, and the
Singapore Ministry of Education (R143-000-411-133 and R143-000-
IV
II
between Pt /Pt within a complex cellular environment.
Koide and Garner developed a fluorescein-based chemo-
dosimeter that detects Pt through Tsuji–Trost allylic bond
512-112).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201305734.
[
16]
cleavage. The system required prior conversion of the Pt
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0
species into Pt , but was capable of detecting cisplatin with
high specificity in a range of buffer conditions. Using
combinatorial library synthesis, Tae et al. also disclosed
II
a rhodamine–triazole probe that is capable of detecting Pt
species such as cisplatin through the ring opening of its
[
17]
spirolactam motif.
We reasoned that it would be possible to engineer
a fluorescent probe specifically designed for the detection of
II
Pt drugs in a cellular environment by taking advantage of the
IV
II
difference in reactivity between the Pt prodrug and its Pt
IV
congener. As Pt prodrug complexes are kinetically inert
because of their saturated coordination sphere and stable
electronic configuration, we could design functional groups
II
that would selectively interact with Pt species. Examples of
such functional groups include dithiocarbamates and dithio-
[
18]
carbamate esters. Indeed, in clinical trials, sodium dieth-
yldithiocarbamate (NaDDTC) was found to sequester excess
administered cisplatin, and to mitigate systemic toxicity
Figure 2. Molecular representation of Rho-DDTC; ellipsoids set at
50% probability.
[
19]
induced by the therapy.
Our strategy was to tether the
non-fluorescent spirolactam form of rhodamine B (RhoB) to
a recognition motif comprised of DDTC (Supporting Infor-
mation, Scheme S1). The recognition motif would bind Pt
II
2+
+
of Pt was high, as the use of other metal ions, such as Na ,
+
2+
2+
2+
2+
2+
3+
species through the dithiocarbonyl bond; thereby, the trans
K , Mg , Cu , Ni , Zn , Fe , and Fe , which play
important roles in biological systems, only led to baseline
values in the emission and absorption spectra (Figure S8).
Compared to Pt , the detection of cisplatin was slower under
similar reaction conditions and required at least 60 min before
a fluorescence signal could be measured. The selectivity for
cisplatin compared to other metal ions remained high, even at
elevated concentrations (Na, K, and Mg at ca. 15 mm), which
is essential for its application as an intracellular probe
(Figure S9).
To fully exploit Rho-DDTC as a probe for Pt drugs, we
sought to understand its underlying mechanism of action. We
postulated that aquation was essential for probe activation,
because cisplatin readily undergoes aquation to form reactive
aqua species. However, even after aquation was suppressed
with 1m NaCl, the probe could still be activated, which thus
suggests alternate pathways. Hence, we evaluated Rho-
DDTC against several cis-[PtA X ] scaffolds (A =
am(m)ine) to understand the role of the ligands in probe
activation (Table S2). Intriguingly, when chelating A ligands
were present, the Pt complex could not activate the probe.
To understand this observation, the reaction products of
K PtCl , cisplatin, carboplatin, and JM118 with Rho-DDTC
were analyzed by ESI-MS. The mass shifts indicated retention
of an ammine and a cyclohexylamine ligand on the Rho-
DDTC/cisplatin and Rho-DDTC/JM118 adducts, respec-
tively (Figure S10). JM118 exclusively formed adducts con-
taining the cyclohexylamine ligand (m/z 1057), whereas
adducts with the alternate ammine ligand (m/z 976) were
absent. Furthermore, the same adducts were formed from
cisplatin and carboplatin, which indicates that CBDCA was
displaced.
ligands are labilized for reactions with the spirolactam motif,
[
20]
and the fluorescence is selectively turned on.
2
+
The target probe Rho-DDTC was prepared in three steps
(
Scheme S1). RhoB was treated sequentially with POCl and
3
NH OH to obtain hydroxamate derivative 1. Reaction with
2
excess 1,2-dibromoethane in the presence of triethylamine as
a base yielded 2, which was quantitatively converted into
Rho-DDTC by a reaction with NaDDTC. The C2 spacer
between the Rho and DDTC motifs was employed to
facilitate positioning of bound Pt at the spirolactam upon
1
DDTC-binding. Rho-DDTC was fully characterized by H
1
3
and C NMR, ESI-MS, and single-crystal X-ray crystallo-
graphic analyses (see the Supporting Information). The spiro
form was confirmed in solution by the presence of the
1
3
C resonance of the spiro C atom at ca. 65 ppm (CDCl and
3
[
D ]DMSO), as well as in the solid state (Figure 2, C8). The
6
crystal structure shows that the thiocarbonyl group is oriented
towards the spirolactam, presumably to minimize steric
2
2
repulsion between the NEt substituent of the carbamate
2
II
and the xanthine ring, thus affording a suitable binding
configuration.
2
+
Detection of Pt using Rho-DDTC was investigated in
HEPES buffer (20 mm, pH 7.4, 30% EtOH). The probe was
stable over a large pH range (3.5–12) and hence compatible
with physiological conditions (Figure S4). When K PtCl was
2
4
2
4
2
+
added as the Pt source (80 mm), a visible change from
colorless to pink was observed after 5 min with a 60-fold
increase in absorbance at lmax = 565 nm, as observed by UV/
Vis spectroscopy (Figure S5). A strong fluorescence turn-on
response was also recorded at lem = 584 nm (l = 490 nm)
ex
2
+
with a 65-fold enhancement relative to the control. At Pt
concentrations between 10–80 mm, both absorbance and
fluorescence increased linearly (Figure S6). Analysis of
a Jobꢀs plot indicated maximum fluorescence intensity at
a mole fraction of 0.5, which suggests that Rho-DDTC
Taken together, the data suggested that the displacement
of an am(m)ine ligand is a critical step for Rho-DDTC
activation. We reasoned that, in the absence of aquation, Pt
complexes first bind to a reactive DDTC motif with
concomitant displacement of an ammine ligand (Scheme S2).
II
2
+
interacted with Pt in a 1:1 ratio (Figure S7). The selectivity
2
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The incident thiocarbonyl group labilizes X because of the
trans effect; this induces nucleophilic attack on Pt by the
spirolactam motif and activates the probe. As initial binding
of JM118 would be affected by steric hindrance from cyclo-
hexylamine, we expect the ammine ligand to be displaced.
Furthermore, the chelating ethylenediamine (en) ligand
cisplatin was observed between two and ten times its IC50
value (Figure S11) and dispersed throughout the cytoplasm.
We noted that the fluorescence did not co-localize with the
nucleus stain because Rho-DDTC was unable to penetrate
the nuclear compartment. We therefore applied our fluores-
II
cence detection system to image Pt complexes (30 mm) in
confers kinetic stability to cis-[Pt(en)Cl ], making displace-
HeLa cells to validate the observations. Intracellular fluores-
cence turn-on was achieved for cisplatin and JM118, which is
consistent with the earlier selectivity tests (Figure 3; see also
Figure S12). Oxaliplatin, which did not activate Rho-DDTC
in buffer solution, was not observed in treated cells under
these conditions (Figure S12). These tests demonstrated that
the detection of cis-[PtA X ] scaffolds by Rho-DDTC did not
2
ment of the amine ligand unfavorable. Indeed, for oxaliplatin,
probe activation was not observed, presumably because of its
stable DACH chelate.
We therefore investigated the ability of Rho-DDTC to
II
IV
distinguish between Pt drugs and their parental Pt com-
IV
plexes. In contrast to cisplatin, Pt complexes 3–6, which are
2
2
based on the cisplatin template, did not trigger fluorescence
turn-on (Figure 3). The reduction of Pt prodrug complexes
arise from non-specific interactions within the complex
cellular environment.
IV
IV
The current understanding entails that anticancer Pt
prodrugs are themselves inactive and require chemical
II
reduction to cytotoxic Pt species, although this has never
[
22]
been directly observed in live cells.
This evidence was
partially provided by a XANES study on frozen pellets of
IV
A2780 ovarian cancer cells that had been treated with Pt
prodrug complexes at high concentrations (200 mm), which
II
IV
quantitated a high proportion of Pt species relative to Pt
after 2 h of exposure.
[
10b,23]
We speculated that this rapid
reduction occurs after cell entry within the cytoplasm.
IV
Complexes 3 and 6 were selected as representative Pt
prodrug complexes at opposite ends of the activity spectrum.
Complex 6 is hydrophobic (logP = 1.01), whereas 3 is highly
hydrophilic (logP = À2.81). Although both complexes are
stable in culture media for 72 h, 3 resisted reduction by virtue
[6d,23]
of its high reduction potential.
Cells treated with 3 and 6 (30 mm) exhibited fluorescence
II
after 3 h of treatment, indicating fast conversion into Pt
species (Figure 4). Fluorescence in cells treated with 3 was
Figure 3. Fluorescence profile of Rho-DDTC (l =490 nm) after treat-
ex
II
IV
ment with Pt and Pt complexes in HEPES buffer.
is highly dependent on the nature of their axial ligands.
Previous structure-activity relationship (SAR) studies have
shown that complexes with axial chloride ligands are most
readily reduced, followed by carboxylates, with hydroxo
[5a,21]
ligands being most resistant.
Rho-DDTC was robust
enough to distinguish 4 from cisplatin. Similarly, Rho-DDTC
could distinguish JM118 from satraplatin, both of which
contain the asymmetric am(m)ine pharmacophore, thereby
providing a direct way of investigating this clinically impor-
tant drug candidate in biological systems.
We exposed HeLa cells to varying concentrations of
cisplatin, from 0.5 to 10 times its IC50 value (3 mm) for 3 h to
establish the sensitivity of the probe. Treated live cells were
incubated with Rho-DDTC, fixed with 4% paraformalde-
hyde, stained with Hoechst 33342 nuclear dye, and imaged
under a confocal fluorescence microscope. Internalization of
Figure 4. Merged fluorescence images of HeLa cells. a) Untreated
control), and exposed to b) cisplatin, c) 3, and d) 6 (30 mm) for 3 h
and further incubated with Rho-DDTC (30 mm) for 1 h at 378C.
(
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well-dispersed within the cytoplasm, which is in line with the
results obtained for cisplatin, but at lower levels. In contrast,
the fluorescence profile observed for cells treated with 6 was
more intense and concentrated around the outer nuclear
envelope. This was presumably due to the highly hydrophobic
nature of 6, which results in its accumulation at the nuclear
lipid bilayer and is the subject of ongoing investigations. The
strong fluorescence profile of cells treated with 6 instead of 3
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[
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[
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complexes within live cells. The probe is compatible with
II
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Received: July 3, 2013
Published online: && &&, &&&&
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Communications
Communications
Cellular Imaging
Watching closely: A fluorescent probe
was engineered to detect the clinically
relevant platinum drug cisplatin within
a complex cellular environment, thus
providing a direct means for visualizing
its cell entry and the activation of plati-
num(IV) prodrugs in cancer cells.
D. Montagner, S. Q. Yap,
W. H. Ang*
&&&& — &&&&
A Fluorescent Probe for Investigating the
Activation of Anticancer Platinum(IV)
Prodrugs Based on the Cisplatin Scaffold
6
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ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 6
These are not the final page numbers!