Pt(IV) prodrugs designed to bind non-covalently to human serum albumin for drug delivery

Article abstract of DOI:10.1021/ja5038269

Albumin is the most abundant protein in human serum and drugs that are administered intravenously inevitably interact with it. We present here a series of platinum(IV) prodrugs designed specifically to enhance interaction with human serum albumin (HSA) for drug delivery. This goal is achieved by asymmetrically functionalizing the axial ligands of the prodrug so as to mimic the overall features of a fatty acid. Systematic variation of the length of the aliphatic tail tunes the cellular uptake and, consequently, the cytotoxicity of cis,cis,trans-[Pt(NH₃)₂Cl₂(O ₂CCH₂CH₂COOH)(OCONHR)], 4, where R is a linear alkyl group. Investigation of an analogue bearing a fluorophore conjugated to the succinate ligand confirmed that these compounds are reduced by biological reductants with loss of the axial ligands. Intracellular release of cisplatin from 4 was further confirmed by observing the characteristic effects of cisplatin on the cell cycle and morphology following treatment with the prodrug. The most potent member of series 4, for which R is a hexadecyl chain, interacts with HSA in a 1:1 stoichiometry to form the platinum-protein complex 7. The interaction is non-covalent and extraction with octanol completely removes the prodrug from an aqueous solution of HSA. Construct 7 is robust and can be isolated following fast protein liquid chromatography. The nature of the tight interaction was investigated computationally, and these studies suggest that the prodrug is buried below the surface of the protein. Consequently, complexation to HSA is able to reduce the rate of reduction of the prodrug by ascorbate. The lead compound from series 4 also exhibited significant stability in whole human blood, attributed to its interaction with HSA. This favorable redox profile, in conjunction with the established nonimmunogenicity, biocompatibility, and enhanced tumor accumulation of HSA, produces a system that holds significant therapeutic potential.

Full text of DOI:10.1021/ja5038269

Article
pubs.acs.org/JACS
Pt(IV) Prodrugs Designed to Bind Non-Covalently to Human Serum
Albumin for Drug Delivery
Yao-Rong Zheng, Kogularamanan Suntharalingam, Timothy C. Johnstone, Hyunsuk Yoo, Wei Lin,
Jamar G. Brooks, and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: Albumin is the most abundant protein in human serum
and drugs that are administered intravenously inevitably interact with
it. We present here a series of platinum(IV) prodrugs designed
specifically to enhance interaction with human serum albumin (HSA)
for drug delivery. This goal is achieved by asymmetrically
functionalizing the axial ligands of the prodrug so as to mimic the
overall features of a fatty acid. Systematic variation of the length of the
aliphatic tail tunes the cellular uptake and, consequently, the
cytotoxicity of cis,cis,trans-[Pt(NH3)2Cl2(O2CCH2CH2COOH)-
(OCONHR)], 4, where R is a linear alkyl group. Investigation of
an analogue bearing a fluorophore conjugated to the succinate ligand
confirmed that these compounds are reduced by biological reductants
with loss of the axial ligands. Intracellular release of cisplatin from 4
was further confirmed by observing the characteristic effects of cisplatin on the cell cycle and morphology following treatment
with the prodrug. The most potent member of series 4, for which R is a hexadecyl chain, interacts with HSA in a 1:1
stoichiometry to form the platinum-protein complex 7. The interaction is non-covalent and extraction with octanol completely
removes the prodrug from an aqueous solution of HSA. Construct 7 is robust and can be isolated following fast protein liquid
chromatography. The nature of the tight interaction was investigated computationally, and these studies suggest that the prodrug
is buried below the surface of the protein. Consequently, complexation to HSA is able to reduce the rate of reduction of the
prodrug by ascorbate. The lead compound from series 4 also exhibited significant stability in whole human blood, attributed to its
interaction with HSA. This favorable redox profile, in conjunction with the established nonimmunogenicity, biocompatibility, and
enhanced tumor accumulation of HSA, produces a system that holds significant therapeutic potential.
metal center. The resulting pseudo-octahedral Pt(IV) complex
is more inert to ligand substitution than the parent Pt(II)
complex and consequently reduces the extent of platinum
sequestration and deactivation en route to the tumor cell.
Within the reducing environment of the cancer cell, the Pt(IV)
center is converted to Pt(II) with release of two ligands and
regeneration of the active square-planar Pt(II) complex. Owing
to the activation step required for Pt(IV) complexes, they
generally exhibit less potency and toxicity than their Pt(II)
counterparts. In addition to kinetic inertness, one of the great
advantages of the Pt(IV) prodrug approach is that the axial
ligands can be used to tune the physical and chemical
properties of the complex without altering the structure of
the active pharmacophore that is ultimately released. In this
way, axial ligands have been used to tune the lipophilicity and
redox potential of Pt(IV) complexes with the aim of altering
cellular uptake and the kinetics of reductive activation.11−14 In
an alternative approach, bioactive ligands can be attached to the
axial positions of a Pt(IV) complex to impart targeting15,16 or
INTRODUCTION
■
The FDA-approved platinum-based drugs cisplatin, carboplatin,
and oxaliplatin are widely used in the clinical treatment of
cancer.1,2 Cisplatin and carboplatin are extensively employed to
treat ovarian, lung, head, and neck cancers, whereas oxaliplatin
is marketed for the treatment of colorectal cancer.3 The
anticancer activity of these compounds arises from their ability
to form intra- and interstrand cross-links on DNA via
coordination of the N7 atoms of purine bases to the platinum
center.1,4 One of the prominent effects of these cross-links is
inhibition of DNA and RNA polymerases, which, when stalled,
can initiate a signaling cascade that leads to cell death.5 Only a
small portion of the platinum administered to a patient
platinates the DNA of cancer cells, and reaction with off-target
nucleophiles leads to toxic side effects including nephrotoxicity,
myelosuppression, peripheral neuropathy, ototoxicity, and
nausea.2,6
One strategy to improve the therapeutic index and decrease
side effects that has received a significant amount of attention is
the Pt(IV) prodrug approach.7−10 Pt(IV) prodrugs are typically
prepared by chemical oxidation of an active square-planar
Pt(II) species, which adds two so-called “axial” ligands to the
Received: April 16, 2014
Published: June 6, 2014
© 2014 American Chemical Society
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additional biological activity.17−21 Axial ligands can also be
chosen to facilitate delivery of platinum complexes by a
nanosized object. Hydrophobic carboxylates have proved
valuable for delivery of platinum complexes within the core
of nanoparticles formed from amphiphilic block copoly-
mers22−25 or within carbon nanotubes.26 A significant impedi-
ment to the clinical progression of Pt(IV) prodrugs is
premature reduction in the bloodstream,27,28 and nanodelivery
constructs such as those described above may help protect the
platinum center as it travels to the tumor.
In this paper, we present the development of a series of
Pt(IV) complexes designed to exploit an endogenous protein,
human serum albumin (HSA), as a delivery device. A key
aspect in our design was the generation of compounds that
mimic the amphiphilic structure of fatty acids. This goal was
accomplished by preparing an asymmetrically functionalized
cisplatin prodrug in which one axial ligand is succinate and the
other an unbranched aliphatic carbamate (Scheme 1). We
as a carrier of anticancer drugs can be attributed to its
nonimmunotoxicity, long circulation time in blood, and high
tumor accumulation.34,35 Although the interaction of platinum
anticancer agents with HSA has been extensively inves-
tigated,36−38 only recently have systems been designed to
exploit HSA as a vehicle for delivering platinum agents. In one
instance, a covalent bond was formed between the pendant
maleimide of a Pt(IV) complex and the sole free cysteine thiol
of HSA.39
We begin with an investigation of the effect of systematically
increasing the chain length of the carbamate ligand of 4a−e on
cellular uptake and cytotoxicity. The most promising
compound, 4e, was investigated further for its ability to interact
with HSA. The resulting non-covalent protein-platinum
complex is highly robust and can be purified by fast protein
liquid chromatography (FPLC), permitting its physical and
biological properties to be examined. Finally, the stability of 4e
in blood was investigated by exploiting the change in
lipophilicity that occurs on reduction from Pt(IV) to Pt(II).
The results of these studies indicate that 4e is orders of
magnitude more stable in blood than previously investigated
Pt(II) and Pt(IV) compounds and that its ability to interact
with HSA may confer this stability.
Scheme 1. Synthetic Route for Preparing 4a−e from
cisplatin: (a) 30% H2O2, 50 °C; (b) succinic anhydride,
anhydrous DMSO, R.T.; (c) R−NCO, anhydrous DMF,
R.T
RESULTS
■
Synthesis and Characterization. A series of amphiphilic
Pt(IV) constructs was prepared using the synthetic approach
shown in Scheme 1. Hydrogen peroxide oxidation of cisplatin
(1) in water affords 2. The asymmetrically functionalized
Pt(IV) compound 3 can be obtained by reaction of 2 with
succinic anhydride.40 The fortuitous acetone solubility of the
disuccinate compound and the insolubility of 3 provide an
effective means of removing the undesired side product.41 The
ability of isocyanate reagents to undergo nucleophilic attack by
the platinum-bound hydroxide ligand of 342,43 was exploited to
form a series of amphiphilic Pt(IV) carbamate species. In this
series, compounds 4a−e bear a hydrophobic unbranched
aliphatic chain varying in length from C2 to C16. The pendant
carboxylate of the succinate ligand trans to the carbamate was
used for further functionalization as described below. The
Pt(IV) compounds were fully characterized by multinuclear
(1H, 13C, and 195Pt) NMR spectroscopy and electrospray
ionization mass spectrometry (ESI-MS) (Figures S1−S5 in the
hypothesized that this structural motif would facilitate
association of 4 with HSA. HSA is the single most abundant
protein in human blood, present at concentrations of 34−54 g
L−1,29 and it serves a wide variety of functions ranging from the
maintenance of blood colloidal osmotic pressure to the
transport of hormones and bilirubin to the buffering of pH.
One of the other important functions it serves is the transport
of fatty acids in the blood. These molecules typically exhibit
very low aqueous solubility, but association with HSA allows
them to be solubilized up to 2 mM.30 Serum albumin has also
been widely investigated for its ability to interact with drug
molecules that are administered intravenously. This interaction
has been exploited in Abraxane, an FDA-approved oncology
product that uses HSA to deliver paclitaxel. It is currently
marketed for the treatment of non-small cell lung cancer, breast
cancer, and pancreatic cancer.31 Clinical trials are currently
underway for a series of similar constructs, currently known as
ABI-008 through ABI-010, which deliver docetaxel, rapamycin,
and tanespimycin, respectively.32 A recent investigation
demonstrated that cancer vaccines can also be tethered to
HSA, allowing delivery to lymph nodes.33 The success of HSA
1
Supporting Information). In the H NMR spectra of 4a−e, an
increase in the intensity of the resonant absorption from δ =
1.2−1.3 ppm arose due to the increasing number of methylene
units in the carbamate chain. Across the series, little change was
observed in the peaks at 2.3, 2.9, 6.5, and 6.6 ppm arising from
the succinate CH2CH2 fragment, carbamate CH2NHCO
fragment, and ammine ligands. These compounds all display
a
195Pt NMR signal around δ = 1240 ppm, confirming the 4+
oxidation state of the platinum. In the ESI mass spectra, well-
defined isotopic distribution patterns provided further con-
firmation of chemical composition. Chemical purity was
established with combustion analysis and analytical HPLC
(Figure S6 in the Supporting Information). The latter also
provides information about the increase in hydrophobicity on
transitioning from 4a to 4e. The compounds show systemati-
cally increasing retention on a C18 reverse-phase stationary
phase as the length of the chain increases. The lipophilicity of
the compounds can be directly evaluated by measuring the
extent to which they partition between octanol and water, Po/w
or simply P. Measured log P values are reported in Figure S8 in
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Figure 1. Cytotoxicity profiles of the Pt(IV) prodrugs: (a) table of measured IC50 values for 4a−e and 7 in A549, A2780, and A2780CP70 cell lines;
(b) calcein AM/ethidium homodimer-1 cell viability assay, details of which may be found in the main text and Supporting Information (white scale
bar, 20 μm; blue scale bar, 200 μm).
the Supporting Information. As expected, the experimental log
P values increased from 4a (log P = −1.72 0.21) to 4e (log P
= 1.23 0.04). The log P values of these complexes were also
calculated using the online program ALOGSP 2.143.44 The
calculated log P values are linearly proportional to the
experimentally measured log P values (Figure S8 in the
Supporting Information).
which is about 8 times higher than that in the A2780 ovarian
cancer cell lines (IC50 = 0.16 0.04 μM).
Cellular Uptake. The extent of cellular uptake was
investigated by treating A2780 ovarian cancer cells with 5 μM
of cisplatin or one of 4a−e for 5 h. The whole cell
concentration of platinum was then evaluated by graphite
furnace atomic absorption spectroscopy (GFAAS). As
expected, the results (Figure S11 in the Supporting
Information) indicate that increase in chain length from C2,
4a, to C16, 4e, significantly enhances cellular uptake. The lead
compound, 4e, is taken up by the A2780 cells 160 times more
effectively than 4a and 40 times better than cisplatin. Notably,
from 4a to 4e, the 160-fold increase in cellular uptake is
mirrored by a 280-fold increase in cytotoxicity, suggesting that
the increase in cytotoxicity can be attributed in large part to the
increase in uptake. The subcellular distribution of 4e was
investigated and revealed that most of the platinum is present
in the cytosol, indicating that, despite its lipophilic character, it
does not become trapped in the membrane (Figure S12 in the
Supporting Information).
Activation by Reduction. Given that Pt(IV) prodrugs are
posited to undergo obligatory reduction prior to anticancer
activity, we investigated the reduction of a cisplatin prodrug
bearing axial succinate and carbamate ligands. Reduction of 5, a
fluorescent analogue of 4, was evaluated using fluorescence
spectroscopy (Figures S15−S17 in the Supporting Informa-
tion). Compound 5 has a quantum yield (ϕ) of 0.047, and the
succinyl-coumarin ligand alone, 6, has a ϕ of 0.85 (Figure S16
in the Supporting Information). The emission of these two
compounds is depicted in Figure S16b in the Supporting
Information. Upon excitation at 365 nm, 5 exhibits no visible
emission, whereas 6 has intense blue emission. Compound 5
shows no significant turn-on upon standing in PBS even after
overnight incubation at 37 °C. When a 10 μM solution of 5 was
treated with 10 equiv of ascorbic acid in PBS, the coumarin
ligand began to detach within 4 h, as confirmed by analytical
HPLC and fluorescence spectroscopy (Figure S17 in the
Supporting Information).
To study the reduction of this class of compounds, a
coumarin molecule was attached to the pendant carboxylate of
4b using standard amide-bond-formation chemistry to produce
5. As with 4a−e, multinuclear NMR and mass spectra are
consistent with the formulation of the compound as shown in
Figure S9 in the Supporting Information. The photophysical
and chemical properties of this compound are discussed below.
Cytotoxicity Profiles. The in vitro anticancer activity of
these newly synthesized Pt(IV) compounds was assessed by
using the MTT assay. Three human cancer cell lines, namely,
the non-small cell lung cancer cell line A549, ovarian cancer cell
line A2780, and cisplatin-resistant ovarian cancer cell line
A2780CP70, were evaluated. Cancer cells were treated with
cisplatin or one of 4a−e for 72 h and cell viability was
evaluated. IC50 values, which represent the concentration
required to inhibit growth by 50%, are given in Figure 1a.
The in vitro anticancer activity of 4e, the most potent member
of the series, was also confirmed by using fluorescent
microscopy and the LIVE/DEAD cell assay, a combination of
the ethidium homodimer-1 assay and staining with acetome-
thoxycalcein, or calcein AM (Figure 1b). Live cells stain with
calcein AM and yield a green fluorescence signal, whereas dead
cells exhibit no fluorescence or a red signal due to the ethidium
homodimer-1. A2780 ovarian cancer cells treated with 10 μM
4e for 48 h were mostly dead, but those treated with cisplatin
under the same conditions had mostly survived. The IC50 for
cisplatin at 48 h is 6.35 1.39 μM, and that for 4e is 0.26
0.04 μM. These cytotoxicity data agree with the observation in
LIVE/DEAD cell assay. In addition, we further evaluated the
cytoxicity of 4e in normal human cells. The IC50 value of 4e in
the MRC-5 (normal lung tissue) cell line is 1.34 0.13 μM,
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Figure 2. DNA damage and cellular responses of A2780 cells treated with 4e: (a) immunostaining of the biomarker of DNA damage, phosphorylated
H2AX (γH2AX) (scale bar, 20 μm); (b) flow cytometric analysis of cell cycle indicating that 4e induced cell cycle arrest at the G2/M phase at 0.2
μM; (c) 4e can inhibit proliferation in A2780 cells at 0.2 μM; (d) Annexin V/PI coupled flow cytometric analysis showing 4e induced apoptosis in a
large population of cells at 0.2 μM; (e) cellular images show characteristic apoptotic cell changes (blebbing, chromatin condensation, and nuclear
fragmentation) upon treatment with 1 μM of 4e for 24 h (scale bar, 20 μm).
Figure 3. Systematic investigation of the Pt-HSA complex, 7: (a) schematic representation of the formation of 7; (b) Job plot of the Pt-HSA
complex showing 1:1 stoichiometry; (c) FPLC trace of 7 and the corresponding GFAAS trace indicate that 4e is tightly bound to HSA; (d) dialysis
experiment indicating that around 60% of the platinum compound was released from 7 over 72 h and that the HSA is retained within the 3 kDa
MWCO micro-dialysis bag; (e) ESI-mass spectrum of the octanol extract showing an intense signal at m/z = 700.1 for [4e − H]− supporting the
integrity of 4e in 7; (f) octanol extraction analysis showing that >90% of 4e (3 μM) was reduced by ascorbate (30 μM) after 2 h incubation at 37 °C
in the absence of HSA. The reduction was decreased to <50% by forming the Pt-HSA complex (3 μM); (g) cytotoxicity profile of 7 against A2780
human ovarian cancer cells showing an IC50 value of 0.12 0.01 μM.
DNA Binding. DNA is the typical cellular target of cisplatin,
and so the ability of 4 to platinate nuclear DNA was assessed.
Five million A2780 cells were treated with growth medium
containing 5 μM cisplatin, 4a, or 4e for 5 h, followed by
incubation in fresh media for an additional 16 h. The
intracellular DNA was isolated and the quantity of bound
platinum was measured by GFAAS. The extent of DNA
platination (Figure S18 in the Supporting Information) was
determined to be 255 55 Pt adducts/106 nucleotides for 4e,
36.4 5.5 Pt adducts/106 nucleotides for cisplatin, and 31.2
4.1 Pt adducts/106 nucleotides for 4a.
Biomarker of DNA Damage Caused by 4e. γH2AX, the
phosphorylated form of histone protein H2AX, is a known
biomarker of DNA damage caused by cisplatin.45,46 γH2AX can
be detected by immunoblotting and immunostaining. As shown
in Figure 2a, fluorescence microscopy can be used to visualize
the localization of γH2AX in the nucleus of A2780 cells treated
with 4e. Similarly, the phosphorylation of H2AX in A2780 cells
treated with 4e could also be observed by Western blotting
(Figure S19 in the Supporting Information), indicative of
genomic DNA damage.
Cellular Responses. DNA-flow cytometric studies were
conducted to identify the incidence of cell cycle arrest following
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Figure 4. Docking studies of 4e and HSA using Autodock Vina: (a) lowest energy structure; (b) platinum complex is buried beneath the protein
surface; (c) platinum compound interacting with a variety of amino acid residues.
treatment with 4e. As shown in Figure 2b, 4e arrests the cycle
in A2780 cells in a time-dependent manner. After 24 h
incubation, a large proportion (66.5%) of cells accumulated at
the S (51.1%) and G2/M (15.4%) phases. After 72 h
incubation, cells were arrested at G2/M (96.5% of whole cell
population). Cell proliferation experiments provide further
evidence for the induction of cell cycle arrest by 4e. As shown
in Figure 2c, 4e completely inhibits proliferation in A2780
cancer cells at concentrations as low as 0.2 μM. Using a dual
staining annexin V/PI flow cytometry assay, the occurrence of
apoptosis was studied in A2780 cells treated with 4e (Figure 2d
and Figure S20 in the Supporting Information). The results
show that 4e can efficiently induce apoptosis in A2780 cells
after 72 h. Even at a low concentration of 0.2 μM, 4e prompts a
large population of cells to undergo early (55.9%) and late
(9.07%) stage apoptosis. Apoptotic cells normally exhibit
changes in cell morphology, such as blebbing, chromatin
condensation, and nuclear fragmentation, all of which can be
observed by fluorescence microscopy. A2780 cells treated with
4e display a dose- and time-dependent morphological change
(Figure S21 in the Supporting Information). As shown in
Figure 2e, blebbing occurs following treatment of A2780 cells
with 1 μM 4e for 24 h. Chromatin condensation and nuclear
fragmentation was also identified by Hoechst staining. All these
cell-based experiments clearly indicate that 4e can effectively
induce DNA damage and thus lead to cell cycle arrest and
apoptosis in cancer cells.
Interaction with Human Serum Albumin. Owing to the
general similarity of the amphiphilic compounds 4a−e to fatty
acids, we hypothesized that they may be able to interact with
HSA and take advantage of this association for transport in the
blood (Figure 3a). The binding affinity of HSA (Sigma-Aldrich,
A1887) toward 4a, 4d, and 4e was investigated by using
fluorescence spectroscopy. Specifically, fluorescence quenching
of the Trp214 residue caused by the Pt(IV) complexes was
measured (Figure S22 in the Supporting Information). The
compound bearing a C16 hydrophobic chain, 4e, displayed the
highest affinity, with a binding constant (Ka) of 1.04 × 106 M−1.
Compound 4d exhibited weaker binding (Ka = 3.7 × 104 M−1),
whereas 4a bound poorly (Ka = 2.6 × 103 M−1). Analysis of a
Scatchard plot (Figure S22 in the Supporting Information)
revealed a 1:1 binding ratio between 4e and HSA, further
supported by a Job plot (Figure 3b). In PBS, the concentration
of 4e at saturation is 3 μM, but upon complexation with HSA
to form 7, Pt concentrations of up to 400 μM can be achieved.
An FPLC analysis was carried out to characterize 7. The
features of the UV−vis trace of 7 are similar to those of pure
HSA, implying that binding of 4e leads to minimal structural
changes in the protein (Figure S23 in the Supporting
Information). Moreover, the features of the Pt trace of 7,
representing the Pt content in the fractions of effluent as
measured by GFAAS, match well those of the UV−vis trace
(Figure 3c), suggesting that 4e is tightly bound to HSA. A
comparison of the UV−vis and GFAAS data reveals that 4e and
HSA associate in a 1.1:1 ratio to form 7. This result is
consistent with the 1:1 ratio obtained from the fluorescence
studies (vide supra). Release of 4e from 7 was evaluated by
dialysis of a PBS solution of the construct against water. A 10
μM solution of 7 in PBS was loaded into a 3 kDa MWCO
micro-dialysis bag and, as shown in Figure 3d, ∼60% of the
platinum compound was released after 72 h at room
temperature. The nonpolar nature of 4e permits facile
extraction of the complex from aqueous solutions into octanol.
Over 90% of the platinum compound can be extracted from an
aqueous solution of 7 in this manner, as determined by GFAAS
(Figure S24 in the Supporting Information). The molecular
identity of the species extracted into octanol was confirmed by
ESI-MS (Figure 3e).
Reduction of 4e and 7. Upon reduction, the hydrophobic
Pt(IV) compound 4e (log P = 1.23) produces a hydrophilic
Pt(II) product, cisplatin (log P = −2.19), which is scarcely
extracted into octanol. Therefore, by measuring the Pt content
in an octanol extract of a mixture of products of reduction of
4e, the degree of reduction can be established. Reduction of 4e
by ascorbate was evaluated using this method. Two different
concentrations (30 μM and 300 μM) of ascorbate were used,
corresponding to the concentrations found in blood and the
intracellular environment, respectively. Compound 4e (3 μM)
is readily reduced by ascorbate at both low and high
concentrations (Figure 3f and Figure S25 in the Supporting
Information). After 2 h incubation at 37 °C, >90% of 4e is
reduced. In a separate experiment, a 3 μM solution of 7
exhibited much slower reduction of the Pt(IV) species after
similar treatment. Even after 7 is incubated with reductant for 5
h (Figure 3f), more than 50% of the platinum remains as the
hydrophobic Pt(IV) species.
Cytotoxicty of 7. MTT assays were carried out to assess
the cytotoxicity of 7 (Figures 1a and 3g). Complex 7 isolated by
FPLC exhibits comparable cytotoxicity to 4e in ovarian cancer
cell lines A2780 and A2780CP70 (IC50 = 0.12
0.01 μM
against A2780 and IC50 = 0.13 0.01 μM against
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A2780CP70). These data represent a 60-fold improvement in
efficacy over that of cisplatin.
amphiphilic nature due to the presence of a long hydrophobic
chain and a terminal carboxylic acid (Scheme 1). Compounds
4a−e were readily synthesized by employing validated chemical
reactions, providing access to this class of previously
unreported Pt(IV) prodrugs bearing trans carbamate and
carboxylate axial ligands.
Molecular Modeling. Docking studies were conducted to
further investigate the non-covalent interaction between 4e and
HSA to form 7. The HSA protein scaffold from 1E7H was used
in a broad search for interactions of 4e. Of the top nine
strongest interactions found, six place the platinum complex in
the same pocket. This pocket, located in subdomain IIA, is
known as Sudlow’s site I and has been previously identified as a
locus of drug interaction with HSA.47,48 The lowest energy
structure is shown in Figure 4a. The platinum complex is buried
beneath the protein surface (Figure 4b). The more polar
fragments of the molecule, which lie closer to the platinum
center, interact with a variety of amino acid residues (Figure
4c). Specifically, Arg233 interacts with the pendant carboxylate
of the succinate. Glu292, Glu153, and His288 are in close
proximity to the ammine ligands bound to the Pt. Arg257 is
directed toward the carbamate carbonyl, and Gln169 is
positioned above the carbamate fragment. The lipophilic C16
tail is coiled into a hydophobic channel lined by Tyr150,
Leu238, Leu219, Leu234, Phe223, Ile264, Ala261, Ile290, and
Tyr150. The Pt(IV) center is located approximately 1 nm away
from Trp214, allowing for quenching of the fluorescence from
the tryptophan residue, which is consistent with the
fluorescence studies described above.
Stability of the 4e in Whole Human Blood. The stability
of Pt(IV) prodrugs in blood is critically important for their
application in clinical settings. We therefore investigated the
stability of 4e in whole human blood by exploiting the ability of
octanol to selectively extract unreduced 4e from aqueous
solution. Compound 4e (60 μM) was incubated in fresh whole
human blood at 37 °C. After 0, 1, 2, 4, and 7 h, aliquots were
extracted with octanol to remove the remaining Pt(IV)
complex. The Pt content in the octanol extract was measured
using GFAAS, and the structural integrity of the extracted
material was confirmed using analytical HPLC. As shown in
Figure 5, the Pt content of the octanol extract decreases to 49%
The lipophilicity of this set of compounds, directly evaluated
by measuring log P, could be readily tuned simply by increasing
the length of the aliphatic chain of the carbamate ligand. An
increase in the length of the chain, and consequently in the
lipophilicity, led to a systematic increase in cytotoxicity from 1
order of magnitude lower to almost 2 orders of magnitude
higher than that of cisplatin.50 This phenomenon was observed
in all three of the cell lines tested (Figure 1a). The lead
compound of the series, 4e, which bears a C16 hydrophobic
chain, exhibited the most potent in vitro anticancer activity. In
all three of the cell lines tested, the cytotoxicity of 4e is
substantially greater (9−70 times) than that of cisplatin (Figure
1a). As shown in Figure 1a, 4e also displays a lower resistance
factor in ovarian cancer cell lines than cisplatin or 4a−d. The
activity of 4e is demonstrated by staining cells with calcein AM
and ethidium, the so-called LIVE/DEAD cell assay. In this
assay, treatment with cisplatin or 4e at equimolar concen-
trations led to drastically different degrees of cell survival.
A likely source of this increased potency lies in the greater
ability of lipophilic compounds to traverse the plasma
membrane. As expected, cellular uptake of the platinum
compounds 4 by A2780 cancer cells increases as the length
of the carbamate chain increases. As suggested in a recent
report, cellular uptake of Pt(IV) compounds is highly related to
their in vitro activity.51 Plotting log [IC50 (μM)] vs log [uptake
(pmol Pt/million cells)], reveals the linear relationship between
the two (R2 = 0.98) depicted in Figure 6a. A plot of log [uptake
(pmol Pt/million cells)] vs log P (Figure 6b) also exhibits a
Figure 5. Stability of 4e in whole human blood showing a half-life of
6.8 h.
after 7 h incubation. HPLC analysis (Figure S26 in the
Supporting Information) indicates that the extracted Pt species
is indeed 4e by comparison with pure material. This result
reveals the half-life of 4e in fresh whole human blood to be 6.8
h.
DISCUSSION
■
Figure 6. Structure−activity relationship of 4a−e between hydro-
phobicity (log P), cytotoxicity (IC50), and cellular uptake in A2780
cells: (a) 2D correlation of cytotoxicity of the platinum(IV) complexes
and their cellular uptake (b) 2D correlation of cellular uptake and
hydrophobicity; (c) 2D correlation of hydrophobicity and cytotoxicity;
(d) 3D plot of hydrophobicity, cytotoxicity, and cellular uptake (black
squares). The projections shown in the various planes are the
corresponding 2D plots (parts a−c).
Pt(IV) Prodrugs with Similarity to Fatty Acids. It can be
appreciated that the structures of 4a−e bear an overall likeness
to those of fatty acids. Although molecular similarity can be
rigorously defined,49 the metrics typically employed are
unsuitable in this particular instance given that there is no
meaningful set of dissimilar compounds with which to contrast
this similarity. Like fatty acids, 4a−e have an overall
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linear relationship (R2 = 0.99). The anticipated linear
relationship also occurs between log P and log [IC50 (μM)]
(Figure 6c), confirming that hydrophobicity directly contrib-
utes to the cytotoxicity of these complexes. Using these three
factors, a 3D structure−activity plot was constructed (Figure
6d). The graph clearly establishes a three-way relationship
between hydrophobicity, cellular uptake, and cytotoxicity.
Mechanism of Action. After entry into cells, Pt(IV)
prodrugs are activated by reduction.10 Reaction of 4a−e with
intracellular reductants such as glutathione or ascorbic acid will
release cisplatin, which subsequently conveys anticancer
activity. Use of fluorescence methods to detect the reduction
of Pt(IV) species in biological environments has attracted much
recent attention.52−55 In order to investigate the redox behavior
of 4a−e, we prepared a coumarin-conjugated Pt(IV)
compound, 5, which displays a significant increase in
fluorescence intensity upon reduction. The similarity between
the coordination spheres and 195Pt NMR chemical shifts of 4a−
e and 5 indicate that the metal-centered redox properties of the
latter are relevant in assessing those of the former. In 5, the
fluorescence of the coumarin fluorophore is quenched owing to
the heavy atom effect induced by platinum. Upon reduction,
the coumarin-containing ligand is released, as determined by
HPLC, resulting in enhanced fluorescence. Using 5, it was
possible to ascertain that Pt(IV) constructs of the type 4a−e
can be reduced by biologically relevant concentrations of
ascorbate within hours. Following such reductive activation,
4a−e exert their anticancer activity via the released cisplatin.
This active Pt(II) moiety induces cell death by forming DNA
cross-links. Accordingly, DNA from A2780 cells that had been
treated with 4a−e contained amounts of platinum roughly
proportional to the cytotoxicity of the compound, and
phorphorylation of H2AX, a biomarker of DNA damage, was
detected in the same cell line following treatment with 4e. The
mechanism of action of 4a−e was further probed by analyzing
the influence of treatment with the platinum agent on the
progression of cells through the cell cycle. Platination is
recognized by the cell as DNA damage, which induces cell cycle
arrest. If the damage cannot be repaired, cells are signaled to
commit apoptosis. The observation of G2/M arrest following S
phase arrest is consistent with the known response of cells to
cisplatin treatment.1 Cisplatin activates the G2/M checkpoint,
allowing DNA to be repaired before mitosis and at the same
time preventing cisplatin-damaged DNA from being inherited
by daughter cells. The induction of apoptosis was confirmed by
the annexin V/propidium iodide (PI) assay. In healthy cells,
phosphatidylserine (PS) is located on the cytoplasmic surface
of the cell membrane. In apoptotic cells, PS is translocated from
the inner to the outer surface of the plasma membrane,
exposing PS to the external cellular environment where it can
be detected by annexin V conjugates. Combining annexin V
and PI, both early and late stage apoptosis can be identified.
Indeed, 4e, the most potent member of the series, induces a
large population of cells to enter into early and late stage
apoptosis. The morphological changes that are expected to
accompany apoptosis, such as blebbing and chromatin
condensation, were also observed.
approaches it via association with HSA. The quenching is
complete within a few seconds of adding the platinum complex,
before acquisition of the first fluorescence measurement. This
phenomenon can be used to create a Job plot, which reveals
that 4e associates with HSA in a 1:1 stoichiometry. Isolation of
the protein by FPLC and quantification of the amount of
protein and platinum in this species confirms the 1:1 molar
ratio. The nature of the interaction between the two is
proposed to be non-covalent because extraction of an aqueous
solution of 7 with octanol removes 4e from HSA in a nearly
quantitative manner. ESI-MS measurements on the octanol
extract confirm the molecular identity of 4e in the non-polar
phase.
The 1:1 stoichiometry implies that a specific non-covalent
interaction may be occurring between 4e and HSA. Molecular
docking simulations were used to investigate possible binding
sites for 4e on the protein. The lowest energy binding site
coincides with Sudlow’s site I.47,48 This pocket has been
classically associated with drug binding and is one of the sites to
which fatty acids bind.56 As can be seen in Figure 4, the
platinum complex is buried beneath the protein surface when
bound in this pocket. This feature suggests that association with
HSA may slow biological reduction of the Pt(IV) center by
preventing reductants from forming an activated complex with
the anticancer prodrug.
The influence of HSA complexation on the reduction of 4e
was probed by exploiting the high lipophilicity of this platinum
complex. As described above, extraction of an aqueous solution
of 7 is able to completely remove 4e from the protein.
Compound 4e was added to whole human blood and incubated
at 37 °C. The platinum complex rapidly associates with HSA to
form 7 in situ. At various time points an aliquot of the
platinated blood was extracted with octanol. The Pt(IV) species
with its axial lipophilic carbamate is extracted into the octanol,
whereas the hydrophilic cisplatin, produced by reduction, is
not. Quantification of the platinum extracted into octanol at
each time point therefore provides a measure of the rate of
reduction in blood. HPLC analysis of the octanol phase
confirms that 4e is the extracted species. The half-life of 4e in
blood obtained from these measurements is 6.8 h, which is
significantly greater than that of cisplatin (t1/2 = 21.6 min) or
satraplatin (t1/2 = 6.3 min).28,57 These favorable chemical
properties combine with the inherent nonimmunogenicity,
biocompatibility, and enhanced tumor accumulation of HSA to
produce a system that holds significant therapeutic potential.
Rapid association between 4e and HSA, in conjunction with
the high concentration of the latter in blood, suggests that
injection of 4e as a small molecule agent will result in the rapid
in situ formation of 7. We envisage, however, that for practical
reasons 4e would be better formulated as 7 and administered as
the protein-platinum complex. This approach permits much
higher concentrations of 4e to be solubilized in water and
eliminates the need to administer 4e using organic cosolvents
or additives.
CONCLUSION
■
In conclusion, we presented the successful development of a
novel Pt(IV) prodrug, 4e, that is capable of associating with
serum albumin to form a Pt:HSA construct, 7. Compound 4e
was obtained from the study of a series of amphiphilic Pt(IV)
complexes designed to mimic the structure of fatty acids. By
tuning length of the hydrophobic chain present in these
compounds, cytotoxicity could be modulated from an order
Interaction with HSA. The amphiphilic structure of
compounds 4a−e mimics that of the fatty acids that are
naturally transported by HSA. We therefore investigated the
interaction of the most potent complex, 4e, with this protein.
The fluorescence of Trp214, the sole tryptophan in the protein,
is quenched by the heavy atom effect when a platinum complex
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found, 644.1. Anal. Calcd for C17H37Cl2N3O6Pt: C, 31.63; H, 5.78; N,
6.51. Found: C, 31.48; H, 5.25; N, 6.40.
Synthesis of 4e. Reagents used in the reaction: Compound 3 (50
magnitude lower to almost 2 orders of magnitude greater than
that of cisplatin. The lead compound, 4e, exhibits excellent in
vitro anticancer activity, showing 9−70 times better activity
than cisplatin in lung and ovarian cancer cell lines. Investigation
of the fluorophore-bearing anolog, 5, confirmed that the
prodrugs described here interact with biological reductants, lose
axial ligands on conversion to Pt(II), and subsequently platinate
DNA. As a consequence of the DNA damage, cell cycle arrest
and apoptosis were observed. A strong non-covalent interaction
between 4e and HSA permits the formation and isolation of 7,
which can act as a delivery vehicle for 4e. This interaction with
HSA protects 4e in the reducing environment of the blood.
This interaction contributes to the promising stability of 4e in
whole human blood. 4e shows a 6.8 h half-life in whole human
mg, 0.117 mmol), hexadecyl isocyanate (69 mg, 0.258 mmol). Yield:
1
32.7 mg (39.9%). H NMR (400 MHz, DMSO-d6): δ: 6.51 (m, 7H,
NH3 and NHcarbamate), 2.87 (q, 2H, CH2(CH2)14CH3), 2.35 (m, 4H,
Succinate), 1.23 (m, 28H, CH2(CH2)14CH3), 0.85 (t, 3H,
CH2(CH2)14CH3).13C NMR (100 MHz, DMSO-d6): δ: 180.0,
174.4, 164.4, 41.5, 31.8, 31.0, 30.4, 30.3, 29.5, 29.5, 29.2, 26.9, 22.6,
14.5. 195Pt NMR (86 MHz, DMSO-d6): 1240.4. ESI-MS (negative
mode) C21H45Cl2N3O6Pt: [M − H]−, calcd, m/z 700.2; found, 700.1.
Anal. Calcd for C21H45Cl2N3O6Pt: C, 35.95; H, 6.46; N, 5.99. Found:
C, 35.72; H, 6.02; N, 5.61.
ASSOCIATED CONTENT
* Supporting Information
■
S
blood, which is significantly longer than that of cisplatin (t1/2
20 min) or satraplatin (t1/2 ∼ 6 min).
∼
Experimental details regarding the characterization of 4a−e and
5, cell culture, the MTT assay, fluorescence studies, FPLC
analysis, immunoblotting, immunostaining, molecular model-
ing, and blood stability tests. This material is available free of
charge via the Internet at http://pubs.acs.org.
MATERIALS AND METHODS
■
General Procedure for the Synthesis of Compounds 4.
Compound 3 was placed into a 5 mL glass vial and an anhydrous DMF
solution (2 mL) of the corresponding isocyanate was added. The
suspension was stirred overnight. The next day, the suspension had
become a clear green solution. The solution was then filtered and the
solvent was removed under reduced pressure at 65 °C. Ethyl ether (2
mL) was added to the oily residue, and the mixture was ultrasonicated
for 1 min and centrifuged. The solid was further washed with DCM (4
mL) and diethyl ether (2 mL). The washed solid was then left under
vacuum overnight.
AUTHOR INFORMATION
Corresponding Author
lippard@mit.edu
■
Notes
The authors declare the following competing financial
interest(s): S.J.L. has a financial interest in Blend Therapeutics.
Synthesis of 4a. Reagents used in the reaction: Compound 3 (200
mg, 0.468 mmol), ethyl isocyanate (70 mg, 0.945 mmol). Yield: 162
ACKNOWLEDGMENTS
■
1
mg (68.6%). H NMR (400 MHz, DMSO-d6): δ: 6.51 (m, 7H, NH3
This work was supported by National Cancer Institute Grant
CA34992. We also thank the Kathy and Curt Marble Cancer
Research Fund (Y.-R.Z. and S.J.L.), a Misrock Postdoctoral
Fellowship to K.S., CCNE, the Samsung Foundation of Culture
for a Samsung Scholarship and the CCNE graduate support for
the CCNE grant (5 U54 CA151884) to H.Y., and MIT UROP
Office support to J.G.B. Spectroscopic instrumentation at the
MIT DCIF is maintained with funding from NIH Grant
1S10RR13886-01.
and NHcarbamate), 2.89 (q, 2H, CH2CH3), 2.34 (m, 4H, succinate),
0.94 (t, 3H, CH2CH3). 13C NMR (100 MHz, DMSO-d6): δ: 180.2,
174.3, 164.2, 36.0, 31.0, 30.4, 15.9. 195Pt NMR (86 MHz, DMSO-d6):
1239.8. ESI-MS (negative mode) for C7H17Cl2N3O6Pt: [M − H]−,
calcd m/z 504.0; found, 503.9. Anal. Calcd for C7H17Cl2N3O6Pt: C,
16.64; H, 3.39; N, 8.32. Found: C, 17.08; H, 3.27; N, 7.98.
Synthesis of 4b. Reagents used in the reaction: Compound 3 (50
mg, 0.117 mmol), hexyl isocyanate (30 mg, 0.236 mmol). Yield: 37.6
1
mg (57%). H NMR (400 MHz, DMSO-d6): δ: 6.51 (m, 7H, NH3
and NHcarbamate), 2.85 (q, 2H, CH2(CH2)4CH3), 2.34 (m, 4H,
succinate), 1.22 (m, 8H, CH2(CH2)4CH3), 0.85 (t, 3H, CH2-
(CH2)4CH3).13C NMR (100 MHz, DMSO-d6): δ: 184.5, 174.4,
164.2, 41.5, 31.6, 30.9, 30.4, 30.3, 26.6, 22.6, 14.4. 195Pt NMR (86
MHz, DMSO-d6): 1242.3. ESI-MS (negative mode) for
C11H25Cl2N3O6Pt: [M − H]−, calcd m/z 560.1; found, 560.0. Anal.
Calcd for C11H25Cl2N3O6Pt: C, 23.54; H, 4.49; N, 7.49. Found: C,
23.52; H, 4.21; N, 7.36.
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