A Potent Glucose-Platinum Conjugate Exploits Glucose Transporters and Preferentially Accumulates in Cancer Cells

Article abstract of DOI:10.1002/anie.201510551

Three rationally designed glucose-platinum conjugates (Glc-Pts) were synthesized and their biological activities evaluated. The Glc-Pts, 1-3, exhibit high levels of cytotoxicity toward a panel of cancer cells. The subcellular target and cellular uptake mechanism of the Glc-Pts were elucidated. For uptake into cells, Glc-Pt 1 exploits both glucose and organic cation transporters, both widely overexpressed in cancer. Compound 1 preferentially accumulates in and annihilates cancer, compared to normal epithelial, cells in vitro. Glucose-platinum conjugates for targeted delivery: A rationally designed potent glucose-platinum conjugate exploits glucose transporters, which are widely overexpressed in cancers, for internalization and selectively accumulates in and annihilates cancer cells.

Full text of DOI:10.1002/anie.201510551

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International Edition: DOI: 10.1002/anie.201510551
German Edition: DOI: 10.1002/ange.201510551
Medicinal Inorganic Chemistry
A Potent Glucose–Platinum Conjugate Exploits Glucose Transporters
and Preferentially Accumulates in Cancer Cells
Malay Patra, Timothy C. Johnstone, Kogularamanan Suntharalingam, and Stephen J. Lippard*
Abstract: Three rationally designed glucose–platinum conju-
gates (Glc–Pts) were synthesized and their biological activities
evaluated. The Glc–Pts, 1–3, exhibit high levels of cytotoxicity
toward a panel of cancer cells. The subcellular target and
cellular uptake mechanism of the Glc–Pts were elucidated. For
uptake into cells, Glc–Pt 1 exploits both glucose and organic
cation transporters, both widely overexpressed in cancer.
Compound 1 preferentially accumulates in and annihilates
cancer, compared to normal epithelial, cells in vitro.
are also overexpressed in certain types of cancer cells.[5,6]
Therefore glycoconjugation becomes an appealing strategy
for targeted delivery of anticancer drugs. The potential of this
strategy in diagnosis and therapy has already been realized,
but there is much room for improvement.[7]
Examples of glucose–platinum conjugates (Glc–Pts) in
which the key structural features of the sugar unit are not
perturbed, a prerequisite for optimal transporter recognition,
and in which the sugar is linked to the platinum complex via
a spacer are scarce.[8] Moreover, these previous studies fail to
answer a crucial question in glycoconjugate development:
Are the conjugates actually taken up by the glucose trans-
porters broadly expressed in cancer cells?
P
latinum-based anticancer drugs are among the most widely
used of all chemotherapeutic treatments. Three FDA-
approved platinum anticancer drugs, cisplatin, carboplatin,
and oxaliplatin, have been in the clinic for many years to treat
a variety of cancers including testicular, ovarian, cervical,
head and neck, non-small-cell lung, and colorectal.[1] Despite
their success, platinum compounds have a number of defi-
ciencies originating from a lack of tumor selectivity. Only
a small fraction of the total administered platinum accumu-
lates at the tumor site, resulting in sub-optimal drug concen-
tration at the target. Moreover, accumulation of platinum in
healthy tissue leads to undesired side effects including
nephrotoxicity, myelosuppression, peripheral neuropathy,
ototoxicity, and nausea.[1b, 2] These drawbacks need to be
addressed when designing next-generation platinum drugs.
Novel strategies for introducing tumor-targeting properties
into platinum anticancer drug candidates are therefore of
great interest.[3]
Herein, we report the synthesis, cytotoxicity, and detailed
characterization of the cellular uptake mechanism of three
novel Glc–Pts 1–3 (Figure 1a). The design of these conjugates
was guided by a recently published crystal structure of the
bacterial xylose transporter XylE, a GLUT1 homolog.[9]
Although a crystal structure of human GLUT1 has also
recently been published,[10] in this study the protein was
captured in the inward open configuration, as opposed to the
outward open configuration that a platinum–glucose conju-
gate would encounter when attempting to enter the cell. The
XylE structure with bound d-glucose, on the other hand,
exhibits the protein in an outward-facing conformation. This
structure reveals that all of the hydroxy groups of d-glucose
except that on C6 are involved in hydrogen-bonding inter-
actions with various amino acid residues of the transporter.
We hypothesized that modification at the C6 position of d-
glucose should not, therefore, interfere with receptor binding.
Previous reports have also suggested that the C6 position of
d-glucose can tolerate various functional groups while
retaining substrate specificity for, and internalization by,
GLUT1.[11] In fact, C6-glucose conjugates of 4-nitrobenzofur-
azan, ketoprofen, and indomethacin were reported to bind
GLUT1 with even higher affinity than unmodified d-gluco-
se.[11a,c,12] This property is highly desirable for a glucose-drug/
fluorophore conjugate, which has to compete with the high
level of glucose (ca. 6 mm) in the blood.[13]
Initial docking studies using a DFT-optimized structure of
the C6-glucose–platinum derivative 1 (Figure 1b and Fig-
ure S17 in the Supporting Information, SI) suggested that this
complex is capable of binding in the cavity of an outward
open XylE. The orientation of the sugar moiety in the docked
complex differs from that of the glucose unit bound in the
crystal structure, but hydrogen-bonding interactions occur
with Gln168, Gln288, Tyr298, and Gln175. These residues had
all been identified as key glucose-binding units in the XylE
structure and either interact directly with the bound d-
glucose or indirectly via hydrogen-bonded water molecules.[9]
In order to maintain cellular homeostasis, growth, and
proliferation, cancer cells significantly increase glucose uptake
and the flux of metabolites through glycolysis. This phenom-
enon, termed “the Warburg effect”, arises from mitochondrial
metabolic changes and is one of the hallmarks of cancer.[4]
GLUT1, the most common glucose transporter, is widely
overexpressed in many human cancers including hepatic,
pancreatic, breast, esophageal, brain, renal, lung, cutaneous,
colorectal, endometrial, ovarian, and cervical.[5] High GLUT1
expression levels in tumor biopsy samples correlate strongly
with poor prognosis. Moreover, several other glucose trans-
porters including GLUT2, GLUT3, GLUT12, and SGLT1/2
[*] Dr. M. Patra, Dr. T. C. Johnstone, Prof. S. J. Lippard
Department of Chemistry, Massachusetts Institute of Technology
Cambridge, MA 02139 (USA)
E-mail: lippard@mit.edu
Dr. K. Suntharalingam
Department of Chemistry, King’s College London
London, SE1 1DB (UK)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201510551.
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The stability of this class of compounds in water and
biological media was evaluated using compound 1. The rate of
activation of platinum drugs by dissociation of the leaving
group ligand(s) from the platinum center in the presence of
biological nucleophiles follows the order dichloride (cispla-
tin) > oxalate (oxaliplatin) > malonate (carboplatin), suggest-
ing high stability for Glc–Pt 1 because its leaving group ligand
is similar to that of carboplatin.[14] Indeed we observed that
1 is highly stable in water as evidenced by no change in the
1H NMR spectrum of 1 after 72 h in D2O (Figure S18). In
RPMI medium, which is used routinely for mammalian tissue
culture, slow activation of 1 by the nucleophiles present in the
medium was observed (Figure S19). This result is consistent
with the previously reported activation of structurally similar
platinum compounds by nucleophiles.[14a,b] No significant
decomposition was noticed up to 8 h, and approximately
60% of 1 remained unchanged even after 24 h, suggesting
that the compound is highly stable in biological media. As
expected, the formation of 5 as a result of activation of 1 was
confirmed by ESI mass spectrometry (Figure S20).
Cellular uptake studies in A2780, DU145 and A549 cells
revealed that, of the three Glc–Pts, 1 is taken up most
efficiently (Figure S5). We also observed
a consistent
decrease in uptake with increasing length of the linker joining
the glucose and platinum moieties. It has been proposed that,
upon binding to the substrate in its outward open conforma-
tion, the GLUT1 transporter undergoes a conformational
change in which the extracellular entrance to the cavity is
occluded and an opening to the cytoplasmic side of the
membrane forms, allowing the substrate to enter into the
cell.[15] Steric hindrance caused by an overly long substrate
could block this conformational change, and we propose that
this phenomenon is responsible for our observation that
glucose–platinum conjugates with longer linkers display
reduced cellular uptake. In this respect, we note that when
the structure of GLUT1 in the inward open form is aligned
with that of XylE in the outward open form into which the
Glc–Pts have been docked, significant steric clashes are
observed for 3 but not 1 (Figure S17).[9,10]
We next evaluated the cytotoxicity of 1–3 and their
aglycone 4 against a panel of human cancer cells of different
origin using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide) assay, a standard assay for cytotox-
icity evaluation. The IC50 (concentration required to reduce
50% cell viability) values derived from dose–response curves
are summarized in Table S1. The cytotox-
Figure 1. a) Structures of Glc–Pts 1–3 and their aglycone 4. b) The
hydrogen-bonding interactions present in the docking model of 1 into
XylE (PDB 4GBZ).[9] The protein is shown as grey ribbons with the
sidechains of key residues depicted as sticks. Complex 1 is shown as
sticks and polar hydrogen atoms are explicitly portrayed. Hydrogen-
bonding interactions are illustrated with dashed lines. (See Figure S23
for a color-coded version of the docking model.)
Additionally, Thr28 is capable of interacting with the
carboxylate ligand of the platinum moiety.
The synthesis of a C6-Glc–Pt compound has not, to our
knowledge, been previously reported. We therefore had to
establish feasible routes to Glc–Pts 1–3 (Scheme 1 and see SI
for details). All new compounds were unambiguously charac-
terized by NMR (1H, 13C, 195Pt) spectroscopy and electrospray
ionization (ESI) mass spectrometry. The purity of the platinum
complexes (1–4) was confirmed to be ꢀ 95% by elemental
microanalyses and analytical HPLC (Figures S1–S4).
icity of the Glc–Pt compounds is generally
comparable to that of aglycone 4, but
greater than that of cisplatin. Ovarian
cancer A2780 cells were the most sensitive
to the Glc–Pt compounds (IC50 = 0.15–
0.22 mm). The relatively tight distribution
of IC50 values for 1–3 suggests that, whereas
the length of the spacer between the
glucose and platinum moieties significantly
influences their cellular uptake, it is not the
primary determinant of the IC50 values of
these compounds.
Scheme 1. Synthetic route for glucose–platinum conjugate 1. Bn=benzyl, DCM=dichloro-
methane, DIPEA=N,N-diisopropylethylamine, TFA=trifluoroacetic acid.
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Although the previously described cellular uptake and
cytotoxicity data may appear inconsistent, it is important to
note that, for technical reasons, these initial assays were
performed on different time scales. We subsequently inves-
tigated the effect of incubation time on the outcomes of these
assays. Whereas the Glc–Pts were designed to be taken up by
facilitated diffusion, the passive diffusion of the aglycone 4
will be impacted significantly by its lipophilicity and conse-
quent ability to traverse the cellular membrane. We found
that, even though the lipophilicity of 4 is approximately one
log P unit higher than that of 1 (Figure S10a), the accumu-
lation of 1 was significantly higher than that of 4 when cells
were incubated with either compound for 8 h (Figure S11).
This result highlights the importance of the glucose moiety of
1 in its cellular uptake. In contrast to the comparable activity
of 1 and 4 observed in the 72 h incubation MTT assay
(Table S1 and Figure S13b), an 8 h incubation MTT assay
revealed 1 to be more cytotoxic than 4 in both A2780 and
DU145 cells (Figures S12 and S13a,b), which is again
consistent with the observed cellular uptake differences
between 1 and 4 in an 8 h assay (Figure S11). We propose
that the initial rate of accumulation of 1 in cells is faster than
that of 4, but that this protein-mediated transport becomes
saturated at longer time scales. On the other hand, the passive
uptake of 4 is slower but does not saturate. As a result,
prolonged incubation with 4 allows the levels of cellular
platinum accumulation and cytotoxicity to approach that of 1.
The difference in the cellular uptake between 1 and 4
diminishes with increased incubation time, monitored from
8 h to 17 h (Figure S13c).
In order to obtain insight into possible subcellular targets of
the Glc–Pts, we studied the intracellular distribution of 1 and 2
as representatives of this class of compound in A2780 cells. As
shown in Figure S6, detection of platinum in the nucleus points
to nuclear DNA as one potential target.[1a] Analysis of DNA
platination levels (Figure S7a) revealed that 1 and 2 platinate
nuclear DNA, the extent of which is 764 Æ57 Pt adducts/104
nucleotides for 1, 483Æ79 Pt adducts/104 nucleotides for 2, and
685Æ17 adducts/104 nucleotides for oxaliplatin, which was
included as a positive control. Increases in the expression levels
of gH2AX, phos-p53, and phos-CHK2, which are canonical
DNA damage biomarkers,[16] were also observed when cells
were treated with increasing concentrations of 1 or 2 (Fig-
ure S7b). As expected for DNA-targeting platinum com-
pounds,[1a] cell cycle arrest at G2/M phase and induction of
apoptosis were observed when A2780 cells were treated with
compounds 1 or 2 and then analyzed by flow cytometry
(Figures S8 and S9). Taken together, these results are consis-
tent with the proposal that Glc–Pts target genomic DNA, the
platination of which leads to apoptosis.
logP values and cellular uptake is consistent with a protein-
mediated transport mechanism (Figure S10a). The ovarian
cancer cell line A2780 was chosen to evaluate the cellular
uptake mechanism of the Glc–Pts because of its high level of
GLUT1 expression,[17] confirmed by immunoblotting analyses
(Figure S10b). Cellular uptake was first monitored in the
absence and presence of an exofacial GLUT1 inhibitor 4,6-O-
ethylidene-a-d-glucose (EDG),[18] and the results are pre-
sented in Figures 2a and S14c. A 50% reduction in cellular
uptake of 1 was measured in the presence of 100 mm EDG.
Under similar conditions, the reduction in the cellular uptake
of 2 and 3 was 38% and 30%, respectively. The cellular
uptake of cisplatin did not change significantly in the presence
of the inhibitor. Because cisplatin can be taken up by passive
diffusion, this result matches well with our expectations. The
inhibitor did, however, cause a 24% decrease in the cellular
uptake of the aglycone 4. Because energy-dependent organic
cation transporters (OCTs) contribute, at least in part, to the
cellular uptake of 4 (Figure 2d, see below), we propose that
the differential uptake induced by the presence of EDG most
likely arises from the energy-depleted conditions produced by
glucose transport inhibition. The extent of cellular uptake
inhibition of the Glc–Pts in the presence of EDG is in the
order 1 > 2 > 3 and this trend tracks with the cellular uptake
of these compounds (Figure S5), providing further support for
the proposal that the GLUT1 translocation efficiencies for
C6-glucose conjugates decrease with increasing linker length.
Cumulatively, these results suggest that the cellular uptake of
1 is not only superior to, but is also more glucose-transporter-
specific than, that of either 2 or 3. As a consequence, only
1 was used in the subsequent cellular uptake experiments.
Similarly to cotreatment with EDG, a 50% reduction in
the cellular uptake of 1 was observed when a structurally and
As described earlier, one crucial question in glycoconju-
gate chemistry is whether or not the sugar-conjugated
molecule is actually transported by the targeted sugar trans-
porters. To address this issue, we carried out a series of
experiments to investigate the details of the mechanism by
which 1–3 are taken up by cells. Glc–Pts 1–3 are very
hydrophilic (logP ꢁ À2) rendering cellular internalization by
passive diffusion through the cellular lipid membrane highly
unlikely. Furthermore, the lack of correlation between the
Figure 2. a) Effect of GLUT1 inhibitor EDG on the cellular uptake of 1–
4 and cisplatin (10 mm compounds, 17 h). b) Effect of externally added
d-glucose and l-glucose (10 mm compounds, 17 h). c) Effect of EDG
on the IC50 values (72 h assay). d) Effect of EDG, Ctd, and their mixture
on the cellular uptake of 1, 4, and oxaliplatin (10 mm compounds, 8 h).
All experiments were done in A2780 cells and cellular uptake in
absence of inhibitor was normalized to 100%. Data represent the
mean ÆSD of at least three replicates. The asterisks denote differ-
ences that are statistically significant (*p<0.01, **p<0.001), ns=not
statistically significant.
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functionally different glucose transport inhibitor, phloretin,
cellular uptake of 1 and indicate that OCT2 facilitates the
was used (Figure S14b). Moreover, given that d-glucose is the
main substrate of GLUT1 and other glucose transporters, it
should compete with and inhibit the protein-mediated uptake
of the Glc–Pts. When probed, d-glucose, but not l-glucose,
exhibited a weak but statistically significant (p < 0.01) inhib-
itory effect on the uptake of 1 (Figure 2b). The poor
inhibitory effect (ca. 30% reduction in uptake) exerted by
d-glucose can be attributed to the high binding affinity of
1 to glucose transporters, a phenomenon previously reported
for other C6-glucose conjugates and GLUT1.[11b,c,12] We also
tested the effect of d-glucose on the cellular uptake of the
aglycone 4 and found the uptake to be unaffected. Further-
more, in cytotoxicity assays carried out in the presence of
EDG, the IC50 value of 1 increased 19-fold (Figure 2c). We
note that EDG does not affect the ability of 1 to platinate
DNA in vitro (Figure S16). In contrast to the results with 1,
only a 6-fold increase in IC50 value was observed during
cotreatment with the control aglycone 4 and EDG. The slight
increase in IC50 value of 4 mirrors the observed decrease in
cellular uptake of 4 in the presence of glucose transport
inhibitors, which we attribute to energy depletion. In order to
probe Glc–Pt uptake through glucose transporters in an
orthogonal manner, we capitalized on the fact that hypoxia
causes stimulation of glucose transport and metabolism in
cancer cells.[19] As shown in Figure S14a, cellular uptake of
1 increased by 50% when A549 cells were treated with the
hypoxia-inducing agent cobalt(II) chloride.[20] No significant
difference in the uptake of 4 was observed under similar
conditions. In summary, the uptake assays support the
hypothesis that glucose transporters, such as GLUT1, are at
least partially involved in the cellular entry mechanism of 1.
It is well documented that organic cation transporter 2
(OCT2) plays important roles in the cellular accumulation
and consequent cytotoxicity of platinum complexes contain-
ing the ((1R,2R)-cyclohexane-1,2-diamine (DACH) ligand.[21]
OCT2-mediated cellular uptake has been suggested as
a leading factor responsible for the sensitivity of colorectal
cancer to oxaliplatin.[21] Because the Glc–Pts reported here
bear the chelating DACH ligand, we investigated the
potential of 1 to undergo translocation via OCT2, a trans-
porter overexpressed in certain types of cancer cells and
tumor samples from patients.[21,22] Expression of OCT2 in
a panel of cancer cell lines was confirmed by Western blotting
analysis (Figure S10b). A2780 cells were incubated with
10 mM 1 for 8 h in the presence or absence of EDG and/or
the OCT2 inhibitor cimetidine (Ctd); oxaliplatin was
employed as a positive control. In the presence of EDG,
uptake of 1 was reduced by 50%, whereas the uptake levels of
4 and oxaliplatin were reduced by only 25% and 30%,
respectively (Figure 2d). The OCT2 inhibitor Ctd reduces the
uptake of the positive control compound oxaliplatin by 70%.
Assays with Ctd revealed reductions of 45% and 35% in the
cellular uptake of 1 and 4, respectively. These results support
the involvement of OCT2 in the cellular internalization of
both 1 and 4. The uptake of 1 was further decreased by 20%
(p < 0.001) following treatment with a mixture of EDG and
Ctd, compared to treatment with Ctd alone. These results
further confirm the involvement of glucose transporters in the
cellular accumulation of 1 as well.
An ideal anticancer compound should be selective for
cancer cells over normal healthy cells, thereby mitigating
undesired toxic side effects associated with chemotherapy. We
therefore evaluated the selectivity of Glc–Pt 1 using DU145
prostate and A498 kidney cancer cells and matched normal
prostate epithelial (RWPE2) and kidney epithelial (CCD1105
KIDTr) cells. Both of the cancerous cell lines have high levels
of GLUT1 expression as compared to the normal epithelial
cells (Figure S10b). Strikingly, as presented in Figure 3, the
accumulation of 1 was significantly higher in the cancer cells
as compared to the matched normal cells. Notably, the cellular
uptake of 1 in DU145 cells is four-fold higher than in RWPE2
cells, and it can be inhibited by the potent glucose transport
inhibitor Cytochalasin B or the OCT2 inhibitor Ctd (Fig-
ure S15), suggesting that the cognate transporters mediate, at
least in part, the preferential uptake of 1 by cancer cells.
Similarly, 1 reduced the viability of cancer cells more
efficiently as compared to normal epithelial cells (Fig-
ure 3c,d).
Given that neuronal cells express high levels of GLUT1
transporters, adverse off-target neurological effects could
arise with these glycoconjugates. We therefore evaluated the
cytotoxicity of 1 in a murine hippocampal-derived Neuro-2A
neuronal cell line, which is known to express the GLUT1
transporter,[23] using short (8 h) and long (72 h) term assays.
As shown in Figure S21, 1 has several-fold higher IC50 values
in Neuro-2A cells (IC50 = (13.6 Æ 0.7) mm and (2.3 Æ 0.3) mm at
8 h and 72 h, respectively) when compared to the most
sensitive overian cancer A2780 cells (IC50 = (2.2 Æ 0.1) mm
and (0.15 Æ 0.06) mm for 8 h and 72 h assays, respectively).
This result indicates that 1 is much more potent in ovarian
cancer cells compared to neuronal Neuro-2A cells in vitro.
Encouragingly, neurotoxicity has not been observed in vivo
for glycoconjugated drugs tested thus far.[7a] Moreover, no
neurological adverse side effects were observed during
a phase II clinical study of glufosamide, a glucose-conjugated
Figure 3. a,b) Preferential accumulation of Glc–Pt 1 in prostate and
kidney cancer cells as compared to matched normal epithelial cells
(20 mm, 8 h). c,d) Effect of Glc–Pt 1 on the viability of cancer and
matched normal cells. The asterisks denote differences that are
statistically significant (*p<0.001, **p<0.02, ***p<0.01,
****p<0.002).
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ifosamide mustard that acts as a DNA alkylating agent.[24]
Finally, we note that the significant body of work showing
preferential accumulation of 18FDG in tumors, used to
diagnose malignancies, underscores the potential of glucose
to become a powerful molecular tag for targeting cancer
cells.[7a,d,25]
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In summary, novel C6-Glc–Pts were rationally designed
and synthesized, and their mechanism of uptake was eval-
uated. Genomic DNA was confirmed to be one of the
intracellular targets of the Glc–Pts. Among the Glc–Pts
investigated, 1 most readily translocates through glucose
transporters. The translocation efficiency and subsequent
cellular accumulation were reduced with increasing size of the
conjugate linker. Strikingly, 1 preferentially accumulates in
and annihilates cancer cells while showing reduced accumu-
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Acknowledgements
This work is supported by the NCI under grant CA034992. We
thank Dr. Samuel G. Awuah for help with flow cytometry.
Keywords: antitumor agents · glucose transporters ·
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How to cite: Angew. Chem. Int. Ed. 2016, 55, 2550–2554
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Received: November 13, 2015
Published online: January 8, 2016
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