Lysosome-Targeting Amplifiers of Reactive Oxygen Species as Anticancer Prodrugs

Article abstract of DOI:10.1002/anie.201706585

Cancer cells produce elevated levels of reactive oxygen species, which has been used to design cancer specific prodrugs. Their activation relies on at least a bimolecular process, in which a prodrug reacts with ROS. However, at low micromolar concentratio

Full text of DOI:10.1002/anie.201706585

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Accepted Article
Title: Lysosome-targeting amplifiers of reactive oxygen species as
anticancer prodrugs
Authors: Andriy Mokhir, Steffen Daum, Viktor Reshetnikov, Miroslav
Sisa, Tetyana Dymych, Maxim D Lootski, Rostyslav Bilyy,
Evgenia Bila, Christina Janko, Christoph Alexiou, Martin
Herrmann, and Leopold Sellner
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201706585
Angew. Chem. 10.1002/ange.201706585
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10.1002/anie.201706585
Angewandte Chemie International Edition
COMMUNICATION
Lysosome-targeting amplifiers of reactive oxygen species as
anticancer prodrugs
Steffen Daum,[a] Viktor Reshetnikov,[a] Miroslav Sisa,[a,b] Tetyana Dumych,[c] Maxim D. Lootsik,[c]
Rostyslav Bilyy,[c] Evgenia Bila,[d] Christina Janko,[e] Christoph Alexiou,[e] Martin Herrmann,[f] Leopold
Sellner[g] and Andriy Mokhir*[a]
Abstract: Cancer cells produce elevated amounts of reactive oxygen
species that has been used to design cancer specific prodrugs. Their
activation relies on at least a bimolecular process, where a prodrug
reacts with ROS. However, at low µM concentrations of the prodrugs
and ROS the activation is usually inefficient. Herein we suggested and
validated a potentially general approach for solving this intrinsic
problem of ROS-dependent prodrugs. In particular, known 4-(N-
e.g. bcr-abl tyrosine kinase and Bruton’s tyrosine kinase (Btk).3
However, cancers, which rely on a single receptor or enzyme, are
rare. Moreover, blood cancers are often genetically
heterogeneous and can evade the action of molecular targeted
drugs due to their intrinsic genetic instability leading to quick
mutations of the target biomolecules.4
More robust targeting can be potentially achieved by prodrug
activation at the cancer specific microenvironment. For example,
the majority of cancer cells overproduce reactive oxygen species
(ROS),5 whereas in non-malignant cells their concentration is
extremely low (0.001-0.7 µM).6 Since elevated ROS seems to be
a general feature of the cancer phenotype, prodrugs activated by
ROS are potentially applicable for the treatment of many different
cancer types.7 Several such prodrugs were reported. In particular,
Jaouen and co-workers have developed ferrocene analogues of
tamoxifen called ferrocifens and their analogues, which are
converted under oxidative conditions into electrophilic products
able to deactivate thioredoxin reductase.8 Jacob and co-workers
have reported on organochalcogenide-based prodrugs, which
catalyze oxidation of glutathione (GSH) to GSSG by H2O2.9 The
group of Peng have introduced pro-alkylating agents, which react
with H2O2 resulting in formation of electrophilic DNA cross-linking
ferrocenyl-N-benzylaminocarbonyloxymethyl)phenylboronic
acid
pinacol ester was converted to its lysosome specific analogue. Since
lysosomes contain the higher concentration of active ROS than
cytoplasm, activation of the latter prodrug was facilitated with respect
to the parent compound. In particular, it was found to exhibit high
anticancer activity in a variety of cancer cell lines (IC50 3.5-7.2 µM)
and in vivo (40 mg/kg, NK/Ly murine model), but remained weakly
toxic towards non-malignant cells (IC50 15-30 µM).
The mode of action of the majority of chemotherapeutic drugs
clinically approved for the treatment of hematologic cancers
including alkylating agents, antimetabolites, anthracyclines and
alkaloids relies on targeting quickly growing cells. However, apart
from cancer cells, some types of non-malignant cells also exhibit
such a property. Therefore, though great progress has been
achieved in this field over the last decades,1 the cancer-cell
specificity of chemotherapeutics is still low and side effects are
dose-limiting. This problem can be approached by using targeted
drugs including either monoclonal antibodies (e.g. alemtuzumab,
ofatumumab)2 or low molecular weight inhibitors of enzymes,
which are either cancer specific or overexpressed in cancer cells,
agents.10
Finally,
our
group
has
developed
N-
alkylaminoferrocene-based prodrugs, which are converted to an
ROS amplifier (electron rich ferrocene) and an alkylating agent
(quinone methide) under cancer specific conditions. Both these
reagents act synergistically by increasing oxidative stress in
cancer cells thereby causing their death.11
The intracellular concentration of H2O2 (the most stable ROS,
[H2O2]in) in cancer cells was estimated to be in the low µM range:
e,g, for Jurkat T-cells it is <7 µM,5a whereas concentrations of
-
more reactive ROS (e.g. O2 , HO) are expected to be even lower.
[a]
S. Daum, MSc; Dr.; V. Reshetnikov, MSc; M. Sisa; A. Mokhir, Prof.
Dr. Friedrich-Alexander-University of Erlangen-Nürnberg,
Department of Chemistry and Pharmacy, Organic Chemistry Chair
II, Henkestr. 42, 91054 Erlangen, Germany
The activation of the ROS-dependent prodrugs occurs in at least
a bimolecular process (a drug reacts with an oxidant), whose rate
is strongly dependent upon concentrations of the reagents:
reaction rate (v)= constant(k)*[prodrug][ROS]. Providing the
prodrug is used at concentrations below 10 µM, the product
[prodrug][ROS] is expected to be <10-10 mol/L that means that
only very quick reactions (large k) are suitable for the efficient
oxidative activation of prodrugs. It is an important intrinsic
limitation of the ROS-dependent prodrugs, since such reactions
are usually rare and not well compatible with aqueous buffers.
Arguably, it can be one of the reasons why not a single ROS-
responsive anticancer prodrug has been clinically approved until
now despite their great potential.
Herein we suggest a potentially general solution of this problem
using N-alkylaminoferrocene-based prodrugs as an example. In
particular, we converted a known prodrug 111e to its analogues
(prodrugs 2 and 5, Figure 1), which target lysosomes (LY’s) due
to the presence of an alkylated piperidine fragment. The latter
moiety is protonated in the acidic environment of LY leading to
E-mail: Andriy.Mokhir@fau.de
[b]
[c]
M. Sisa, Dr., Institute of Experimental Botany AS CR, Prague,
Czech Republik
R. Bilyy, Prof. Dr.; T. Dumych, PhD; M.D. Lootsik, Prof. Dr.; Danylo
Halytsky Lviv National Medical University, Pekarska str. 69, 79010
Lviv, Ukraine
[d]
[e]
E.Bila, Dr.; Department of Organic Chemistry, Ivan Franko Lviv
National University, Kyrylo & Mefodiy Str. 6, 79005, Lviv, Ukraine
C. Janko, Dr.; C. Alexiou, Prof. Dr. med., Department of
Otorhinolaryngology, Head and Neck Surgery, Section of
Experimental Oncology and Nanomedicine (SEON), University
Hospital Erlangen, Glückstraße 10a, 91054 Erlangen, Germany
M. Herrmann, Prof. Dr. Dr.; Department of Internal Medicine 3 -
Rheumatology and Immunology, University Hospital
Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg,
91054 Erlangen, Germany
[f]
[g]
L. Sellner, Dr.; Department of Medicine V, University Hospital
Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/anie.201706585
Angewandte Chemie International Edition
COMMUNICATION
0.02 s-1 for a background reaction (Figure 2A). Previously, we
confirmed that the low activity of 1 could be attributed to its
aggregation in aqueous solution that inhibits its interaction with
H2O2.16 Here we found that in contrast to 1, prodrug 2 has lower
propensity towards aggregation (SI) that explains its higher
reactivity towards H2O2.
200
A
B
2
100
50
0
1
1
100
0
No prodrug
added
2
0
50
100
0
25
[Prodrug] / µM
50
Time / min
G0G1 G2
S
C
D
Figure 1. Structures of aminoferrocene-based prodrugs 1, 2 and 5 as well as
the mechanism of their activation in cancer cells (insert). Paths A and B:
conversions of unspecific prodrug 1 to their lysosome-specific analogues 2
(non-fluorogenic) and 5 (fluorogenic).
1
2
100
50
0
70
35
0
trapping of the hybrid. LY was selected as a target, since H2O2
produced by a cancer cell freely enters its LY’s via diffusion. Since,
in contrast to cytoplasm, LY’s do not contain H2O2-degrading
enzymes,12 H2O2 can get accumulated.13 Additionally, these
organelles contain large amounts of loosely bound iron ions,
which catalyze conversion of H2O2 into highly toxic HO.14 Among
all ROS, the latter one is the most efficient oxidizer of N-
alkylaminoferrocene-based prodrugs.11e Thus, the prodrug
activation in this case will be facilitated by three factors: prodrug
accumulation in LY’s, increased concentration of ROS in these
organelles and higher fraction of the most active ROS - HO.
0 10 20 50 10 20 50
0
1
2
3
4
5
[Prodrug] / µM
[2] / µM
Figure 2. A: Increase of the fluorescence intensity (λex = 501 nm, λem = 531 nm)
upon oxidation of 2’,7’-dichlorofluorescin (DCFH, 9.9 µM) by H2O2 (9.9 mM)
either in the presence of prodrugs 1 and 2 (both 49.5 µM) or in their absence.
The time point of the prodrug addition is indicated with a black dashed arrow.
After the initial lagging period, the oxidation reactions reach their maximum rates.
Linear fits of these regions are indicated with blue lines. Other conditions are
given in the SI. B: Effects of prodrugs 1 (filled triangles) and 2 (open circles) on
the viability of BL-2 cells. C: Increase of the mean fluorescence (λex = 488 nm,
λem
= 530 nm, monitored by flow cytometry) of 5-(6-)chloromethyl-2′,7′-
dichlorodihydrofluorescein diacetate (CM-DCFH-DA)-loaded BL-2 cells
incubated with prodrugs 1 and 2 for 2 h. D: Effect of prodrug 2 (incubation for
24 h) on cell cycle of BL-2 cells.
Prodrug
2 was prepared as described in the supporting
information (SI). The LY-targeting was achieved by addition of a
basic aliphatic tertiary amine residue (piperidine) to the parent
structure 1. Such residues act as LY-specific carriers, since they
are protonated in the acidic environment of LY leading to cargo
accumulation within this organelle.15
Encouraged by these data, we studied toxicity of the new prodrug
2 towards representative human blood cancer cell lines derived
from B- (BL-2) and T-cells (Jurkat) (Figure 2B, Table S1). In both
cases we observed that the piperidine derivative 2 is substantially
more toxic than the parent 1: 3.5 + 0.9 vs 26 + 5 (BL-2, p< 0.001)
and 7.2 + 0.1 vs 44 + 2 µM (Jurkat, p< 0.001). In agreement with
these data prodrug 2 induces 7-35 fold stronger oxidative stress
in BL-2 cells than its analogue 1 (Figure 2C). The induction of the
oxidative stress in BL-2 cells by the prodrugs is accompanied by
initial upregulation of the intracellular antioxidant glutathione
([GSH]in, p<0.01) as measured by staining with GSH-specific dye
monobromobimane (MBB, Figure S12). Furthermore, prodrug 2
causes BL-2 cell cycle arrest in G0/G1 phase: 73 + 3 % at [2]= 3-
5 µM vs 48 + 3 % in the absence of any prodrug (Figure 1D). We
also observed the similar effect for prodrug 1 but at the
substantially higher concentrations: G0/G1 59-66 % at [1]= 25-30
µM (Figure S13). Incubation of Jurkat cells with the prodrugs
causes similar changes in [GSH]in (Figure S21), namely
upregulation of GSH for prodrug 2 ≤ 12.5 µM and oxidation of
GSH to GSSG for prodrug 2 ≥ 25 µM (t= 24h). For the latter cell
line we studied the mechanism of prodrug-induced cell death
(Figure S20). In particular, we found that at the low [2] the cells
First, we investigated basic properties of prodrug 2 and compared
them to those of the previously reported prodrug 1.11e In particular,
we found that lipophilicities of 1 and 2 are similar: logP= 4.5 + 0.3
(for 1); 4.7 + 0.1 (for 2). In agreement with these data, they
permeate the cellular membrane of Burkitt lymphoma BL-2 cells
with the same efficiency (SI). By using ESI mass spectrometry we
confirmed that prodrug 2 is converted to its predicted products in
the presence of H2O2 (Figures 1, S4-S6): B-C cleavage product I
(m/z 539.2000, [M+H]+) and N-alkylaminoferrocene II (m/z
388.1594, [M]+). Additionally, minor peaks corresponding to the
product of decomposition of the ferrocene unit in II (m/z 267.1858,
[M+H]+) and the product of mono-alkylation of II with quinone
methide 4 (m/z 494.2018, [M]+) were detected (SI). Thus, the
mechanism of H2O2-induced activation of 2 remains the same as
that of 1.11 In agreement with the latter statement, we observed
that prodrug 2 facilitates conversion of H2O2 to HO. The latter
reaction was followed by HO-mediated oxidation of non-
fluorescent 2’,7’-dichlorofluorescin (DCFH) to fluorescent 2’,7’-
dichlorofluorescein (DCF). Interestingly, we found that the
catalytic efficiency of the new prodrug is ~4-fold higher than that
of the parent prodrug 1: (dF/dt)0= 7.81 s-1 for 2, 1.97 s-1 for 1 and
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10.1002/anie.201706585
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COMMUNICATION
are killed via both apoptosis and necrosis mechanisms, whereas
in the presence of the high [2] necrosis dominates. The cell-kill
could be partially rescued by the addition of N-acetylcystein
(NAC), which indicates that ROS plays is an important
determinant of the mode of action of prodrugs 1 and 2. Based on
these data, we could conclude that the introduction of a piperidine
residue to the structure of 1 dramatically improves the activity of
the prodrug both in cell free settings and in cell lines. Importantly,
the toxicity of the resulting compound against normal, non-cancer
human dermal fibroblast adult (HDFa) cells remains low (IC50= 30
+ 1 µM) relative to that for BL-2 (p<0.001) and Jurkat cells
(p<0.001) (Table S1). Moreover, the new prodrug is highly active
against primary chronic lymphocytic leukemia (CLL) cells (IC50=
2.0 + 1.1 µM), but remains only weakly toxic against normal
mononuclear cells (MNC’s, IC50= 15 + 7 µM, p<0.01), which
contain a fraction of B cells. The latter is an excellent drug toxicity
model, since both CLL- and B-cells are genetically related to each
other.11b
electron donor, PET is not possible and this compound is
fluorescent (Figure S3). Both prodrugs 2 and 5 were found to have
similar lipophilicities and cell membrane permeabilities (SI).
Furthermore, we observed that prodrug 5 is toxic towards BL-2
cells (IC50= 27 + 3 µM). The lower toxicity of 5 with respect to 2
(p< 0.001) was explained by the decreased ROS-generating
ability of 5 (Figure S11), which is caused probably by coumarin-
induced ROS quenching.
A
B
C
20 µm
25 µm
E
F
D
Based on the initial design prodrug 2 was supposed to target LY’s
of cancer cells. To evaluate whether this is the case, we labeled
LY’s in BL-2 cells with acridine orange (AO). AO exhibits orange
fluorescence when accumulated in LY’s. In contrast, it emits in the
green spectral region when bound to RNAs.17 We detected the
LY-specific emission of the cells (FLY) by using flow cytometry (λex
= 488 nm, λem = 690 nm, Figure S14). In the untreated, AO-loaded
cells mean FLY was found to be high: 176 + 5 arbitrary units (a.u.).
The treatment of the cells with prodrug 1 led to only slight
decrease of mean FLY (111 + 6 a.u., p<0.001). In contrast, the
cells treated with prodrug 2 were practically non-fluorescent
(mean FLY = 19 + 1, p<0.001). These data indicate that prodrug 2
causes LY-disruption in BL-2 cells that probably is the main cause
of prodrug 2-induced cell death. We also reproduced this effect in
human prostate DU-145 cells (Figure S15). The latter cells were
selected since they are adherent and, therefore, can be well
studied using fluorescent microscopy. Moreover, DU-145 cells
are known to generate large amounts of ROS18 and are, therefore,
responsive to aminoferrocene-based prodrugs.11c In particular,
we observed that similarly to BL-2 and Jurkat cells, prodrug 2 was
more toxic towards DU-145 cells (IC50= 6.5 + 0.1) than prodrug 1
(IC50= 24.5 + 0.1). By using fluorescence microscopy, we found
that untreated, AO-loaded DU-145 cells contain yellow dots on
the green background, which could be identified as LY’s (Figure
3, image A). The same pattern was observed in the cells treated
with prodrug 1 (image B). In contrast, the cells treated with
prodrug 2 lack yellow dots that confirms the drug-induced LY-
disruption in this case (image C). Accumulation of prodrug 2 by
LY’s could not be studied directly by the previously reported assay
based on the analysis of boronic acid released in cells upon
activation of the prodrugs, due to its insufficient sensitivity.11
Therefore, we introduced an alternative fluorescence-based
assay: for that purpose, an analogue of prodrug 2 (prodrug 5)
containing a coumarin dye was synthesized (SI). In the intact state
prodrug 5 is practically not fluorescent due to the efficient
photoinduced energy transfer (PET) from the ferrocene moiety to
the dye (Figures 1, S3). However, upon its treatment with H2O2 it
is activated with formation of ferrocenium derivative 6+ and
quinone methide 4 as outlined in Figure 1 and experimentally
confirmed by ESI mass spectrometry (Figures S7-S9). Since, in
contrast to the starting prodrug, product 6+ does not contain any
Figure 3. A-B: Acridine orange (AO)-loaded DU-145 cells; no prodrug added
(A); in the presence of podrug 1 (B) or prodrug 2 (C). D-E: DU-145 cells treated
with Lysotracker Red (LTR) and prodrug 5; red channel is LTR specific (D),
green channel is specific for products released from 5 in cells (E); image F is an
overlap of D and E.
Next, we co-incubated prodrug 5 and Lysotracker Red (LTR: a
LY-specific dye) with DU-145 cells and imaged the cells with
fluorescence microscopy. The LTR dye was detected by using
excitation / emission combination of 538-562 / 570-640 nm (red
color in Figure 3D), whereas the activated 5 was detected by
using excitation / emission combination of 335-383 / 420-470 nm
(green color, E). Overlap of two images appears yellow (F) that
indicates that both dyes are fully co-localized. This experiment
confirms unambiguously that prodrug 5 is accumulated in LY’s
and gets activated in these organelles. The LY-disruption was not
observed at the concentrations of the prodrug used in this
experiment. These data were confirmed by flow cytometry (Figure
S17).
Finally, antitumor activity of prodrug 2 was evaluated in murine
Nemeth-Kellner lymphoma (Nk/Ly) model.19 Two preliminary
experiments including 6 and 13 black C57/BL6N mice, in which
the dose of 2 was optimized, and one final experiment with 14
mice (m~25 g) were performed. In particular, the mice were
inoculated subcutaneously with 7.5*105 NK/Ly cells (day 0), that
initiated the growth of tumor according to lymphosarcoma type.
Then either a carrier (DMSO, 30 µL, control group, N=7) or
prodrug 2 dissolved in the carrier (30 µL, dose 40 mg/kg, test
group, N=7) were injected intraperitoneally (i.p.) on days 1, 3, 5,
7, 9, 11, 13, 15. The increase of weight of animals, which
correlates with tumor progression, was monitored. We observed
that prodrug 2 inhibits tumor growth significantly (Figure 4A). On
day 19 the animals were sacrificed, tumors excised, weighed,
fixed and undergone histological analysis. In all animals except of
one in the test group the tumor weight was substantially reduced
with respect to those from the control group (Figure 4B).
Furthermore, histological analysis of lymphosarcoma from the
group treated with DMSO alone exhibited prominent signs of
neoangiogenesis (marked V) and abundant mitoses (Figure C),
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10.1002/anie.201706585
Angewandte Chemie International Edition
COMMUNICATION
whereas the presence of the large number dying cells (both
apoptotic and necrotic) were observed in tumors from the group
treated with prodrug 2 (Figure 4D).
AM thanks German Research Council (DFG, MO1418 / 7-1) for
funding this project. CA and CJ were supported by the DFG
through the Cluster of Excellence Engineering of Advanced
Materials (EAM).
Keywords: Aminoferrocene • Prodrug • Lysosome • Cancer •
Reactive Oxygen Species
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Synthesis of new prodrugs, description of assays and additional
experimental data are provided in the supporting information (SI).
Acknowledgements
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10.1002/anie.201706585
Angewandte Chemie International Edition
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COMMUNICATION
Steffen Daum, Viktor Reshetnikov,
Miroslav Sisa, Tetyana Dymych, Maxim
D. Lootsik, Rostyslav Bilyy, Evgenia
Bila, Christina Janko, Christoph Alexiou,
Martin Herrmann, Leopold Sellner and
Andriy Mokhir*
Lysosome staining
in DU-145 cells
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Lysosome-targeting amplifiers of
reactive oxygen species as
anticancer prodrugs
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