of 29
A Monofunctional Platinum Complex Coordinated to a Rhodium
Metalloinsertor Selectively Binds Mismatched DNA in the Minor
Groove
Alyson G. Weidmann
and
Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA 91125
Abstract
We report the synthesis and characterization of a bimetallic complex derived from a new family of
potent and selective metalloinsertors containing an unusual Rh—O axial coordination. This
complex incorporates a monofunctional platinum center containing only one labile site for
coordination to DNA, rather than two, and coordinates DNA non-classically through adduct
formation in the minor groove. This conjugate displays bifunctional, interdependent binding of
mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA
sequencing experiments revealed that the preferred site of platinum coordination is not the
traditional
N
7-guanine site in the major groove, but rather
N
3-adenine in the minor groove. The
complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-
proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and
triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium
metalloinsertors.
INTRODUCTION
Platinum anticancer agents comprise an essential component in the current repertoire of
chemotherapeutics.
Cis
-diammineplatinum(II) complexes such as cisplatin (Figure 1) and its
derivatives have been extremely successful in the treatment of a variety of cancers but are
also associated with a litany of severe side effects and resistance.
1
5
These side effects arise
primarily as a result of the mechanism by which the platinum complexes function
biologically: slow displacement of
cis
-oriented labile leaving group ligands, such as
chlorides or carboxylate groups, activates the platinum center for the formation of cytotoxic,
covalent adducts with DNA.
1
,
6
Although these complexes preferentially bind the
nucleophilic
N
7 position of consecutive guanine residues to form what are known as 1,2-
intrastrand crosslinks, the nature of the platinum binding is inherently nonspecific and can
target the DNA of non-cancerous cells as well as malignant ones.
2
Additionally, although
DNA is widely considered to be the primary therapeutic target of cisplatin, platinum(II)
complexes possess the ability to react with a number of biological ligands once inside the
*
To whom correspondence should be addressed at jkbarton@caltech.edu.
Supporting Information
Synthesis and supporting figures. This material is available free of charge via the internet at
http://www.pubs.acs.org
HHS Public Access
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. Author manuscript; available in PMC 2016 October 05.
Published in final edited form as:
Inorg Chem
. 2015 October 5; 54(19): 9626–9636. doi:10.1021/acs.inorgchem.5b01722.
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cell, including proteins. A major source of cisplatin resistance, for example, is the chelation
and subsequent inactivation by sulfur-containing molecules, such as glutathione.
7
Indeed, it
is reported that only 1% of intracellular cisplatin reaches the genome.
8
The ability to tune
platinum therapeutics to target specific biomarkers of cancer would be invaluable in the
development of next-generation platinum drugs.
Our laboratory has focused largely on the development of octahedral rhodium(III)
complexes for the targeted therapy of cisplatin-resistant cancers. These complexes
selectively bind thermodynamically destabilized sites, such as base pair mismatches, in
DNA.
9
Mismatches, which arise naturally as a consequence of DNA replication, lead to
cancerous mutations if left uncorrected by the complex of proteins known as the mismatch
repair (MMR) machinery.
10
,
11
As a result, deficiencies in the MMR pathway result in a
buildup of these single base lesions in the genome, leading to several types of cancer. These
malignancies are largely resistant to cisplatin and other classical chemotherapeutics, as
MMR is involved in the recognition and processing of cisplatin-DNA lesions.
12
As a result,
cisplatin generally exhibits enhanced activity in MMR-proficient cells, leading to a buildup
of resistance as malignant cells continue to proliferate.
Our rhodium complexes recognize DNA mismatches not through the formation of covalent
adducts, as with cisplatin therapeutics, but rather through a non-covalent binding mode.
These complexes bind DNA via insertion of a sterically expansive aromatic ligand, such as
5,6-chrysenequinone diimine (chrysi) (Figure 1) into the base stack of the duplex at the
mismatched site. This event occurs from the minor groove, ejecting the thermodynamically
destabilized, mismatched bases from the helix out towards the major groove.
13
18
This
binding mode, termed metalloinsertion, targets 80% of all mismatches with over 1000-fold
specificity in all sequence contexts.
13
More recently, we have demonstrated that these
metalloinsertor complexes also target mismatches within the genome; metalloinsertors
exhibit cytotoxicity preferentially in MMR-deficient cancer cells compared to isogenically-
matched MMR-proficient cells, and this selectivity is correlated with localization to the
nucleus rather than to the mitochondria.
19
23
Rhodium metalloinsertors are a robust class of complexes that offer a promising alternative
for targeting MMR-deficient cancers and circumventing resistance. New generations of
metalloinsertors have exhibited increased potency surpassing that of cisplatin, while still
maintaining selective targeting to MMR-deficiency.
22
,
24
While these compounds are
currently being explored as chemotherapeutic agents, they also hold promise as potential
adjuvants that could confer their unique selectivity onto other therapeutic cargo. Recent
efforts have focused on the development of bimetallic Rh-Pt complexes that bifunctionally
target DNA through both metalloinsertion at mismatched sites as well as through the
formation of covalent platinum crosslinks. Previous iterations of metalloinsertor-platinum
complexes have included the conjugation of a platinum center to the rhodium complex
through its inert amine ligand
25
as well as the temporary attachment of the two metal centers
via the labile platinum leaving group ligand.
26
In our latest efforts to develop selective bifunctional conjugates, we turn to a new family of
metalloinsertor complexes, developed and characterized only in the last two years. Each
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complex in this new generation of metalloinsertors contains an unusual ligand coordination
involving an axial Rh—O bond. This coordination environment involves the bidentate
N,O
-
chelation of a pyridylethanol ligand hinged by a quaternary carbon center, which can be
functionalized with a variety of non-coordinating substituents. The coordination of anionic
oxygen reduces the overall charge of the complex from [3+] to [2+], which raises the pK
a
of
the complex; the protonated chrysi ligand thereby adopts a puckered rather than planar
configuration when bound to DNA. These complexes also exhibit unprecedented potency in
MMR-deficient cells, while maintaining high cell selectivity. It is purported that the unusual
structure of these metalloinsertors alters their DNA binding mode; the buckled chrysi ligand
potentially inserts into the base stack at a mismatch in a side-on configuration, producing a
unique lesion in the genome that may be more readily recognized in MMR-deficient cells.
Their biological properties as well as high functional group tolerance (with respect to the
non-coordinating substituents on the
N,O
coordinating ligand) make these metalloinsertors
promising new scaffolds for conjugate design.
24
Here, we report the first-generation
conjugate derived from this new family. We have synthesized a new bifunctional
metalloinsertor complex, wherein a cisplatin group is attached to the previously
characterized [Rh(chrysi)(phen)(DPE)]
2+
(phen = 1,10-phenanthroline; DPE = 1,1-
di(pyridin-2-yl)ethanol) via coordination to the extraneous pyridine. This conjugate,
[Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
(Figure 1), thereby contains a monochloro platinum
center with only one labile site available for coordination to DNA, distinguishing it from its
doubly coordinating
cis
-platinum (II) predecessors. In this work, we demonstrate that
[Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
preferentially targets platinum to mismatched DNA
in vitro
and as a result forms unusual, nonclassical platinum adducts with adenine in the
minor groove.
EXPERIMENTAL
Materials
Cisplatin and all organic reagents were purchased from Sigma-Aldrich unless otherwise
noted. Commercially available chemicals were used as received without further purification.
RhCl
3
starting material was purchased from Pressure Chemical Co (Pittsburgh, PA). Sep-
pak C
18
solid-phase extraction (SPE) cartridges were purchased from Waters Chemical Co.
(Milford, MA). Media and supplements were purchased from Invitrogen (Carlsbad, CA).
BrdU, antibodies, and buffers were purchased in kit format from Roche Molecular
Biochemical (Mannheim, Germany).
Oligonucleotides were ordered from Integrated DNA Technologies and purified by HPLC
using a C18 reverse-phase column (Varian, Inc; Corona, CA). All HPLC purifications were
carried out on a Hewlett-Packard 1100 HPLC. DNA purity was confirmed by MALDI-TOF
mass spectrometry and quantified by UV-visible spectroscopy (UV-vis) using the extinction
coefficients at 260 nm estimated for single-stranded DNA. UV-vis characterizations were
performed on a Beckmann DU 7400 spectrophotometer. Radiolabeled [
32
P]-ATP was
purchased from MP Biomedicals (Santa Ana, CA).
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Synthesis
The syntheses of chrysene-5,6-dione (chrysi), 1,1-di(pyridin-2-yl)ethanol (DPE), and
[Rh(chrysi)(phen)(DPE)]
2+
were carried out according to published procedures.
22
,
27
,
28
[Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]Cl
3
A 250 ml round bottomed flask was charged with [Rh(chrysi)(phen)(DPE)]TFA
2
(272 mg,
0.28 mmol) (prepared according to literature procedures) and cisplatin (305 mg, 1 mmol,
3.57 equiv) in 100 ml H
2
O. One drop of concentrated HCl was added, and the solution was
stirred at reflux for an additional 48 h. The reaction was hot-filtered through a medium glass
frit and purified by reverse-phase HPLC (85:15:0.1 to 40:60:0.1 H
2
O/MeCN/TFA gradient).
Fractions were pooled and dried
in vacuo
to afford the bimetallic product as a red-brown
solid. To obtain the complex as the chloride salt, [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl]TFA
3
was redissolved in 50 mM HCl
(aq)
and freeze-dried under high vacuum. This process was
repeated three times until the TFA counterion was eliminated. Yield: 60 mg (16% by
HPLC).
1
H NMR (500 MHz, D
2
O):
δ
9.39 (d,
J
= 5.3 Hz, 1H), 8.95 (d,
J
= 8.0 Hz, 1H),
8.90 (d,
J
= 6.7 Hz, 2H), 8.87 – 8.68 (m, 1H), 8.49 – 8.35 (m, 1H), 8.34 – 8.27 (m, 1H), 8.24
– 8.18 (m, 1H), 8.14 – 8.11 (m, 1H), 8.07 (d,
J
= 10.3 Hz, 1H), 8.03 (s, 1H), 8.00 (d,
J
= 6.7
Hz, 2H), 7.96 (d,
J
= 8.0 Hz, 2H), 7.90 (d,
J
= 7.9 Hz, 1H), 7.81 (s, 1H), 7.72 (d,
J
= 7.5 Hz,
1H) 7.68 – 7.60 (m, 1H), 7.57 (s, 1H), 7.52 (t,
J
= 7.5 Hz, 1H), 7.41 (s, 2H), 7.32 (d,
J
= 6.8
Hz, 2H), 7.27 – 7.17 (m, 1H), 7.00 (d,
J
= 7.8 Hz, 1H), 3.66 – 3.59 (m, 3H), 3.54 (dd,
J
=
5.6, 3.5 Hz, 3H), 2.95 (s, 3H). ESI-MS (cation):
m/z
calc 1003.251, obs. 1001.8 (M – 2H
+
).
UV-vis (H
2
O, pH 7.0): 270 nm (134,700 M
−1
cm
−1
), 303 nm (72,400 M
−1
cm
−1
), 442 nm
(19,200 M
−1
cm
−1
), 581 nm (10,600 M
−1
cm
−1
).
Photocleavage Competition Titration
A 29mer DNA hairpin with the sequence 5’-
GGCAGG
CATGGCTTTTTGCCAT
CCCTGCC −3’ (underline denotes the mismatch) was
labeled at the 5’-end with [
32
P]-ATP using polynucleotide kinase (PNK) at 37 °C for 2 h
followed by purification using gel electrophoresis. A small amount of the labeled DNA (less
than 1% of the total amount of DNA) was added to 2 μM DNA in 100 mM NaCl, 20 mM
NaP
i
, pH 7.1 buffer. The DNA hairpin was annealed by heating at 90°C for 10 min and
cooling slowly to ambient temperature over a period of 3 h. Racemic solutions of
[Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
were prepared in Milli-Q water over a range of
concentrations (100 nM – 50 μM). For each sample, 4 μM
rac
-[Rh(bpy)
2
chrysi]Cl
3
(5 μl),
which photocleaves DNA at mismatched sites, 2 μM annealed mismatched duplex DNA (10
μl), and the non-photocleaving competitor complex at various concentrations (5 μl) were
combined to give 1 μM
rac
-[Rh(bpy)
2
chrysi]
3+
, 1 μM duplex DNA, and 50 mM NaCl
(aq)
as
the final concentrations. Samples were irradiated on an Oriel (Darmstadt, Germany) 1000-W
Hg/Xe solar simulator (340–440 nm) for 15 min, incubated at 37 °C for 10 min, and dried
in
vacuo
. The irradiated samples were electrophoresed on a 20% denaturing polyacrylamide
gel and exposed to a phosphor screen. The amounts of DNA in each band were analyzed by
autoradiography and quantitated by phosphorimagery (ImageQuant).
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Binding Constant Determination
To assess the binding of the rhodium subunit of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
at
the CC mismatch, the fraction of cleaved DNA in each lane on the gel was quantified and
expressed as a percentage of the total DNA in each lane and plotted against the log of the
concentration of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
. The data from three independent
titration experiments were each fit to a sigmoidal curve using OriginPro 8.5. The
concentration of rhodium at the inflection point at the curve ([Rh
50%
]) was then used to
solve simultaneous equilibria involving DNA, [Rh(bpy)
2
chrysi]Cl
3
, and [Rh(chrysi)(phen)
(DPE-Pt(NH
3
)
2
Cl)]
3+
in Mathematica 8.0 to obtain the binding constant (K
B
).
Platinum Binding to Mismatched and Well-Matched DNA
A single-stranded DNA oligomer with the sequence 5*’-TTAGGATCAT
CCATATA-3’
(underline denotes the mismatch, asterisk denotes the radiolabel) was labeled at the 5’-end
with [
32
P]-ATP and polynucleotide kinase (PNK) at 37 °C for 2 h. The radiolabeled DNA
was purified by gel electrophoresis and annealed to either its mismatched complement
(containing a CC mismatch) or a fully matched complement strand by heating to 90 °C in
buffer (100 mM NaCl, 20 mM NaP
i
, pH 7.1), followed by slow cooling to ambient
temperature over 2 h, to give a final concentration of 2 μM duplex DNA. Racemic solutions
of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
were prepared in 50 mM NaCl
(aq)
over a range of
concentrations (100 nM – 5 μM). For each sample, 2 μM annealed mismatched duplex DNA
(10 μl) was mixed with [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
at various concentrations
(10 μl) to give 1 μM duplex DNA and 75 mM NaCl
(aq)
as the final concentrations. A “light”
control, (ØRh, ØPt) consisting of 2 μM DNA mixed with 10 μl Milli-Q water, and a “dark”
control (Ø
h
υ
), containing the DNA mixed with the highest concentration of metalloinsertor
without irradiation, were also prepared. The samples were incubated at 37 °C for periods of
1, 3, or 18h to promote the formation of the platinated DNA adducts. After the incubation
period, samples were quenched with 50 μl of 0.1 M NaCl
(aq)
and cooled to 4 °C for 30 min.
Except for the unirradiated controls, samples were irradiated on an Oriel (Darmstadt,
Germany) 1000-W Hg/Xe solar simulator (340–440 nm) for 15 min and dried
in vacuo
. For
DNA footprinting experiments, platinated DNA was precipitated with ethanol and subject to
the appropriate sequencing method. The irradiated samples were electrophoresed on a 20%
denaturing polyacrylamide gel and exposed to a phosphor screen. The amounts of DNA in
each band were analyzed by autoradiography and quantitated by phosphorimagery
(ImageQuant).
Dimethyl Sulfate Footprinting of Platinated DNA
DNA footprinting of guanine by dimethyl sulfate (DMS) was carried out according to
literature procedures.
29
Radiolabeled duplex DNA (well-matched or CC-mismatched; see
above for sequence) was platinated with varying concentrations of [Rh(chrysi)(phen)(DPE-
Pt(NH
3
)
2
Cl)]
3+
(0, 1, or 5 μM) or cisplatin (1 μM) as described above. The platination
reaction was quenched via addition of 0.1 M NaCl
(aq)
(0.1 ml) followed by cooling to 4 °C
for 30 min. Samples were purified by ethanol precipitation and dried
in vacuo
. The samples
were taken up in 5 μl Milli-Q water, diluted with DMS buffer (50 mM sodium cacodylate, 1
mM EDTA, pH 7.5) (190 μl), and 2 mM calf-thymus DNA (4 μl) was added as a carrier
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DNA. Samples were cooled to 0 °C and treated with 5 μl DMS (10% v/v in EtOH, prepared
immediately before use) for 5 min at 25 °C. The reaction was quenched
via
addition of the
DMS stop solution (1.5 M NaOAc, 1 M
β
-mercaptoethanol, 250 μg/ml yeast tRNA) at 0 °C.
Following ethanol precipitation of the DNA, samples were treated with 10% aqueous
piperidine and heated to 90 °C for 30 min. The piperidine was removed
in vacuo
, and
samples were electrophoresed on a 20% denaturing polyacrylamide gel and exposed to a
phosphor screen. The amounts of DNA in each band were analyzed by autoradiography and
quantitated by phosphorimagery (ImageQuant).
Methylation of Platinated DNA with Methyl Methanesulfonate (MMS)
Radiolabeled duplex DNA (well-matched or CC-mismatched; see above for sequence) was
platinated with varying concentrations of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
(0, 1, or 5
μM) or cisplatin (1 μM) as described above. The platination reaction was quenched via
addition of 0.1 M NaCl
(aq)
(0.1 ml) followed by cooling to 4 °C for 30 min. Samples were
purified by ethanol precipitation and dried
in vacuo
. The samples were taken up in 10 μl
Milli-Q water, diluted with Tris-HCl buffer (10 mM Tris-HCl, 5 mM MMS, pH 7.8) (200
μl), and 2 mM calf-thymus DNA (4 μl) was added as a carrier DNA. The DNA methylation
reaction was allowed to occur at ambient temperature for 16h, followed by ethanol
precipitation. Strand breaks in the reacted DNA were generated by heating methylated DNA
in 10 mM Tris-HCl buffer (0.1 ml) at 90 °C for 15 min to depurinate thermally labile
adducts.
30
Following precipitation with ethanol, DNA was then treated with 1 M piperidine
at 90 °C for 30 min. Samples were then dried
in vacuo
, electrophoresed on a 20% denaturing
polyacrylamide gel, and exposed to a phosphor screen. The amounts of DNA in each band
were analyzed by autoradiography and quantitated by phosphorimagery (ImageQuant).
Analysis of Platinated DNA by Mass Spectrometry
Duplex DNA (1 μM well-matched or CC-mismatched; see above for sequence) was
platinated with 5 μM [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
and incubated alongside
unplatinated duplex DNA at 37 °C for 90 min. The reaction was quenched via incubation at
4 °C for 15 min, and the samples were irradiated for 15 min. The DNA was then treated with
1 M piperidine formate at 60 °C for 15 min, precipitated with ethanol at 4 °C, and
depurinated with 1 M piperidine at 90 °C for 15 min. The piperidine was removed
in vacuo
and the dried DNA samples were analyzed by MALDI-TOF mass spectrometry.
Cell Culture
HCT116N (MMR-proficient) and HCT116O (MMR-deficient) cells were grown in RPMI
medium 1640 supplemented with 10% fetal bovine serum, 400 μg/ml Geneticin (G418), 2
mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 units/ml
penicillin, and 100 μg/ml streptomycin. Cells were grown in tissue culture flasks (Corning
Costar, Acton, MA) at 37 °C under a humidified atmosphere (5% CO
2
).
MTT Cytotoxicity Assay
The cytotoxic effects of conjugate [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
, [Rh(chrysi)
(phen)(DPE)]
2+
, and cisplatin were studied
via
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazolium bromide) assay in the MMR-proficient HCT116N and MMR-deficient
HCT116O cell lines.
31
For biological experiments, [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
and cisplatin were prepared in saline solution (20 mM NaCl), and [Rh(chrysi)(phen)
(DPE)]
2+
was dissolved in deionized water. Cells were plated in 96-well plates at 50,000
cells/well and incubated with varying concentrations of metal complex for 72h under
humidified atmosphere. After the incubation period, MTT was added, and the cells were
incubated for an additional 4 h. The resulting formazan crystals were solubilized over a
period of 24 h at 37 °C, 5% CO
2
. Formazan formation was quantified
via
electronic
absorption at 550–600 nm with a reference wavelength of 690 nm. Cell viability is
expressed as a function of formazan formation and normalized to that of untreated cells.
Standard errors were calculated from 5 replicates.
MTT Caspase and PARP Inhibition Assays
The cytotoxic effects of conjugate [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
and cisplatin
were studied
via
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
in the HCT116O and HCT116N cell lines. Cells were plated in 96-well plates at 50,000
cells/well and incubated with 0 or 5 μM of metal complex. For caspase-inhibition assays, Z-
VAD-FMK was added to a final concentration of 35 μM. For poly-ADP ribose polymerase
(PARP) assays, the inhibitor 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2
H
)-isoquinoline
(DPQ) was added to a final concentration of 50 μM. Controls wherein cells were treated
with inhibitor alone in the absence of metal complex were included. Cells were incubated
under humidified atmosphere for 72 h and labeled with MTT for an additional 4 h at 37 °C,
5% CO
2
. The ensuing formazan crystals were dissolved with a lysis buffer (10% SDS in 10
mM HCl) according to the manufacturer’s instructions. MTT reduction to formazan was
quantified by electronic absorption at 570 nm (background: 690 nm), and percent viability
was expressed as the amount of formazan in treated cells compared to that of the untreated
controls.
RESULTS
DNA Binding Studies
The rhodium mismatch recognition and covalent platinum binding of DNA were analyzed
with mismatched and well-matched DNA oligomers on 20% denaturing PAGE gels. Our
laboratory has previously shown that rhodium metalloinsertors bearing
N,O
-coordinating
ligands containing an axial Rh—O bond bind mismatches as both the
Λ
- and Δ-enantiomers
with equal affinity in contrast to earlier chrysi complexes of Rh. As it has been demonstrated
that [Rh(chrysi)(phen)(DPE)]
2+
exhibits similar mismatch DNA binding affinities in both
the racemic and enantiopure form, we carried out DNA binding experiments on racemic
[Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
. It is possible, however, that the addition of the
platinum center increases preferential binding of one stereoisomer over the other. Mismatch
recognition and platinum adduct formation can be visualized simultaneously under the same
conditions (shown in Figure 2), however, platinum binding is optimally observed under
saline conditions (75–100 mM NaCl
(aq)
). While this affords thermodynamic control over the
DNA platination reaction, thereby enhancing selective platination of mismatched DNA, high
salt concentrations make quantification of photocleavage at the mismatched site challenging.
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As a result, metalloinsertion at the mismatch was analyzed separately from platination of
mismatched and well-matched DNA, under aqueous conditions.
Binding Affinity of Rhodium at a CC Mismatch
In vitro
DNA binding studies were performed with racemic aqueous solutions of [Rh(chrysi)
(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
and radiolabeled hairpin DNA containing a CC mismatch with
the sequence 5*
-GGCAGG
CATGGCTTTTTGCCAT
CCCTGCC-3
(underline denotes the
mismatch; asterisk denotes the radiolabel) Single-stranded DNA was labeled at the 5’-end
with [
32
P]-ATP and polynucleotide kinase (PNK) at 37 °C for 2h as described above. The
conjugate was bound with mismatched hairpin DNA at varying concentrations and irradiated
(340–440 nm) for 15 min. Samples were then incubated at 37 °C for 10 min and
electrophoresed on a 20% denaturing PAGE gel. As [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
does not cleave DNA upon irradiation, a competition titration was carried out using
[Rh(bpy)
2
chrysi]
3+
, which does photocleave DNA at the site of a mismatch.
9
The conjugate
inhibits photocleavage by
rac
-[Rh(bpy)
2
chrysi]
3+
at the mismatched site in a dose-
dependent manner (Figure 2); this inhibition indicates that the complex binds specifically to
the mismatch
via
metalloinsertion. Experimental conditions were carried out to minimize
platinum adduct formation, thus limiting interference of covalent platinum binding on the
equilibrium binding constant of the rhodium subunit at the mismatch. Nevertheless, some
platinum binding is observed to occur simultaneously with mismatch binding, as indicated
by the presence of slowly migrating bands located above the unmodified parent band. This
result suggests that the complex is capable of binding mismatched DNA bifunctionally,
through metalloinsertion at the mismatched site as well as the formation of covalent
platinum adducts. The amount of photocleaved DNA was quantified and plotted against the
logarithmic concentration of the complex (log[Rh(DPE)Pt]), and the K
B
value of
[Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
was calculated by solving simultaneous equilibria at
the inflection point of the titration curve (See Figure S2 of the Supporting Information). The
binding affinity of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
for a CC mismatch, under
conditions where platinum coordination is inhibited, was determined to be 4.8 × 10
6
M
−1
,
comparable to that of monomeric metalloinsertors.
20
,
22
,
23
Platination of Mismatched and Well-Matched DNA
The formation of platinum-DNA crosslinks was analyzed
in vitro
via denaturing
polyacrylamide gel electrophoresis. Dissociation of the labile chloride ligand from the
platinum center in solution enables the formation of covalent platinum adducts with DNA.
The reaction between the conjugate and mismatched (CC) and well-matched duplex DNA
oligomers was analyzed as a function of incubation time at 37 °C as well as complex
concentration.
A time-course experiment was used to explore the formation of Pt-DNA adducts with
radiolabeled duplex DNA of the sequence 5*’-TTAGGATCAT
CCATATA-3’ (underline
denotes the site of a CC mismatch, asterisk denotes the radiolabel) annealed with either its
mismatched or fully matched complement strands. Racemic mixtures of [Rh(chrysi)(phen)
(DPE-Pt(NH
3
)
2
Cl)]
3+
(1 μM) and mismatched or well-matched DNA (1 μM) were incubated
in buffer (75 mM NaCl, 10 mM NaP
i
, pH 7.1) at 37 °C for periods of either 1, 3, or 18 h.
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After the incubation period, samples were quenched with 0.1 M NaCl
(aq)
, cooled to 4 °C,
and electrophoresed on a 20% denaturing PAGE gel. Platination of the DNA is indicated by
the appearance of bands with reduced electrophoretic mobility, located above the
unmodified parent bands in the autoradiogram. The resulting autoradiogram is shown in
Figure 3. The [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
conjugate exhibits a preference for
mismatched DNA over fully matched oligomers after 1 and 3 h incubation periods. The 18 h
incubations resulted in complete degradation of the DNA, and the bands could not be
observed above background.
DNA platination was also analyzed in a dose-dependent manner, as can be seen in Figure 3.
Racemic mixtures of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
(0.1 – 5 μM) and mismatched
or well-matched DNA (1 μM) were incubated at 37 °C for 2h and electrophoresed on a 20%
denaturing polyacrylamide gel. The platinum-DNA bands were quantified by
autoradiography, (See Figure S3 of the Supporting Information), revealing a preference for
mismatched DNA at low concentrations (0.1 – 1 μM) of conjugate. At 500 nM [Rh(chrysi)
(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
, 41 ± 5.4% of mismatched duplex contains platinum adducts
versus 25 ± 5.3% of well-matched DNA. Optimal selectivity is achieved at stoichiometric
Pt:DNA (1 μM), with 52 ± 5.1% platinated mismatched DNA versus 36 ± 5.7% (
p
< 0.05 by
unpaired two-tailed
t
test). Not surprisingly, this differential platinum binding diminishes at
high concentrations of the complex, where mismatched and well-matched DNA are
platinated equally; at 5 μM complex, 72% and 70% platination of mismatched and well-
matched DNA is observed, respectively. It would appear as though the formation of
platinum crosslinks is guided at least in part by mismatch recognition by the rhodium
subunit.
Determination of Platinum Binding Site
Given the preferential platination of mismatched DNA over well-matched sequences, we
performed DNA sequencing reactions to probe the site of covalent binding within the
duplex. Typically, the preferential DNA binding site of platinum(II) complexes is the
N
7
position of guanine, a major groove adduct. Methylation of
N
7-guanine by dimethyl sulfate
(DMS) promotes site-selective depurination of these residues.
29
The degree of DMS-
induced guanine cleavage indicates whether platinum is coordinated; uncoordinated
guanines will incur relatively high levels of cleavage upon DMS treatment, while platinated
sites will be protected. Duplex DNA (1 μM) containing a single CC mismatch, as well as a
similarly well-matched sequence was radiolabeled at the 5’-end with [
32
P] and incubated
with either cisplatin (1 μM) or [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
(1 or 5 μM) for 90
min at 37 °C to promote the formation of Pt-DNA adducts; untreated controls of
mismatched and well-matched DNA were also included. The DNA was then purified and
subjected to treatment with 10% DMS, followed by cleavage by piperidine (1 M) and
denaturing gel electrophoresis (20% polyacrylamide).
The resulting autoradiogram is shown in Figure 4. The cleavage products of the two guanine
residues in the radiolabeled strand are indicated by bands of high electrophoretic mobility
located below the unmodified parent bands. For both mismatched and well-matched DNA,
treatment with [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
conjugate does not confer protection
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of the guanine residues from DMS methylation and cleavage. In fact, a marked
increase
in
guanine cleavage product is observed with conjugate-bound DNA at both 1 and 5 μM
treatment, compared to untreated and cisplatin-treated DNA. Furthermore, this increase is
observed for both guanine residues, which occur consecutively in the sequence. However,
the conjugate clearly forms covalent adducts, as is indicated by the presence of slow-
migrating bands located above the unmodified parent bands. The [Rh(chrysi)(phen)(DPE-
Pt(NH
3
)
2
Cl)]
3+
complex does not coordinate at the expected guanine sites; rather, the
alternative platinum binding site likely results in a conformational change to the DNA that
enhances the accessibility of both guanine residues to methylation by DMS.
As the metalloinsertion binding mode occurs from the minor groove, we considered the
possibility that the platinum subunit forms minor groove adducts with DNA. Specifically,
we explored platination of the
N
3 position of adenine, a common binding site for alkylating
agents that target the minor groove due to its nucleophilicity and its relative accessibility.
32
Treatment of double stranded DNA with methyl methanesulfonate (MMS) leads to
methylation of
N
3-adenine as well as, to a lesser extent,
N
7-guanine in the major groove.
30
As with DMS treatment, a decrease in cleavage at methylation sites is indicative of
protection by platinum coordination. Mismatched and well-matched DNA duplexes were
treated with either cisplatin (1 μM) or [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
(1 or 5 μM)
as described above and exposed to MMS (5 mM in 10 mM Tris-HCl buffer, pH 7.8) for 18 h
at ambient temperature. Alkylated DNA was cleaved by heating at 90 °C followed by
treatment with hot piperidine (1 M), and samples were electrophoresed on a 20% denaturing
polyacrylamide gel.
Figure 4 displays the resulting autoradiogram. Note that depurination due to MMS
methylation appears to occur more readily at residues near the ends of the DNA strand and
less so in the middle of the sequence, likely due to accessibility. Quantitated PAGE data are
shown in Figure S4 of the Supporting Information. Near the 3’-end of the radiolabeled
strand, a decrease in cleavage of adenine residues occurs concurrently with the appearance
of slow-migrating Pt-DNA bands. This decrease suggests that platinum coordination confers
protection of these residues from methylation. At the 5’-end of the labeled strand of the
mismatched sequence, an
increase
in cleavage is observed with increasing conjugate
concentration at the adenine and guanine residues located the farthest away from the
mismatched site. This effect is less pronounced for the well-matched sequence (See Figure
S4 of the Supporting Information), suggesting that the duplex distortions that promote
methylation by MMS at these sites in mismatched DNA are perhaps less severe when the
complex is bound to well-matched DNA.
As a corollary to denaturing PAGE experiments, we also analyzed platinated DNA by mass
spectrometry. Mismatched and well-matched sequences were platinated as described
previously and exposed to 1 M piperidine formate at 60 °C for 15 min to depurinate the
DNA. Following purification by ethanol precipitation, samples were cleaved at purine
residues via treatment with 1 M piperidine at 90 °C. Samples were analyzed by MALDI-
TOF mass spectrometry, and the results are summarized in Table 1. The corresponding
spectra are shown in the Supporting Information. Whereas MMS footprinting primarily
labeled purine residues located near the ends of the nucleic acid sequence, mass
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spectrometry analysis herein favors cleavage towards the middle of the sequence.
Nevertheless, fragments corresponding to platinated adenine sites were observed for both
mismatched and well-matched sequences.
MTT Cytotoxicity Assay
The cytotoxic effects of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
were probed via MTT
assay (MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).
Metabolically active cells reduce MTT to formazan, which has a characteristic absorbance at
570 nm. Quantification of formazan by electronic absorption indicates the amount of viable
cells in each sample.
31
The isogenically matched human colorectal carcinoma cell lines
HCT116N (MMR-proficient) and HCT116O (MMR-deficient) cells were plated in 96-well
plates at 5.0 × 10
5
cells/well and treated with varying concentrations of [Rh(chrysi)(phen)
(DPE-Pt(NH
3
)
2
Cl)]
3+
. Cells were also treated with each parent subunit, [Rh(chrysi)(phen)
(DPE)]
2+
and cisplatin, for 72h under humidified atmosphere. Percent viability is defined as
the ratio of the amount of formazan in treated cells to that of untreated cells. The cytotoxic
effects of the complexes in the HCT116N and HCT116O cell lines are shown in Figure 5.
As expected, the [Rh(chrysi)(phen)(DPE)]
2+
parent complex displays cell-selective
cytotoxicity in the MMR-deficient HCT116O line, with an IC
50
value of approximately 3.5
μM. Cisplatin exhibits no effect in either cell line, possibly due to being administered from
saline solution, to provide an adequate control for the [Rh(chrysi)(phen)(DPE-
Pt(NH
3
)
2
Cl)]
3+
complex, which is also prepared in aqueous NaCl (20 mM). The conjugate
displays intermediary cytotoxic effects compared to its monomeric rhodium and platinum
subunits: the cell selectivity of the rhodium subunit is abolished, as both MMR-proficient
and MMR-deficient cell lines are targeted equally. However, the conjugate exhibits
enhanced potency compared to its platinum subunit (IC
50
10 μM), signifying that
conjugation to rhodium does play some role in enhancing the efficacy of the cisplatin parent
complex, either through increased cellular uptake or DNA targeting. The potency of
[Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
is also comparable to the earlier-generation
metalloinsertor-oxaliplatin conjugate, which has an IC
50
value of 9 μM in the HCT116O cell
line.
26
Caspase and PARP Inhibition Assays
Characterization of a previous metalloinsertor-platinum conjugate revealed that the
cytotoxic effects arose not from the necrotic cell death mechanism induced by monomeric
metalloinsertors,
21
but rather through an apoptotic pathway more characteristic of
cis
-
platinum complexes.
26
,
33
,
34
Here, we examined whether [Rh(chrysi)(phen)(DPE-
Pt(NH
3
)
2
Cl)]
3+
also triggers apoptosis, which may account for its lack of cell-selectivity.
HCT116N and HCT116O cells were treated with conjugate (5 μM) and poly-ADP ribose
polymerase (PARP) inhibitor 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2
H
)-isoquinoline
(“DPQ,” 50 μM)
35
for 72h, and cell viability was assayed by MTT. Cells were treated
similarly with DPQ (50 μM) and cisplatin (5 μM) as a control. The addition of PARP
inhibitor DPQ protects cells from necrotic death, as PARP mediates this pathway through
severe depletion of cellular ATP.
36
As can be seen in Figure 6, treatment of both cell lines
with DPQ alone effects no change in viability. Similarly, DPQ has no effect on the viability
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of cells treated with [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
. HCT116N cells exhibit 66 ±
2.0% cell viability in the presence of conjugate alone, and 61 ± 2.8% viability with metal
complex administered in combination with PARP inhibitor. For HCT116O cells, viability is
73 ± 3.4% and 71 ± 2.0% in the presence of the conjugate alone and the combination
treatment, respectively. These results indicate that the cytotoxic effects of [Rh(chrysi)(phen)
(DPE-Pt(NH
3
)
2
Cl)]
3+
in HCT116 cells are independent of the PARP pathway and therefore
do not proceed via necrosis.
Curiously, co-treatment of cells with cisplatin (5 μM) and DPQ (50 μM) results in a
statistically significant increase (
p
< 0.0001 by unpaired two-tailed
t
test) in cell viability
compared to treatment with cisplatin alone: the percentage of viable HCT116N cells
increases from 86 ± 4.3% to 95 ± 1.0% upon the addition of PARP inhibitor, and the
fraction of viable HCT116O cells increases from 73 ± 3.6% to 82 ± 1.1%. While these are
modest changes overall, these results suggest that cisplatin induces necrosis in these cell
lines to some degree.
The experiment was also performed in the presence of a pan-caspase inhibitor, Z-VAD-
FMK. By irreversibly binding to the active site of caspases, Z-VAD-FMK inhibits
apoptosis.
37
Previously, it has been shown that appendage of a platinum moiety to a
metalloinsertor triggers caspase-dependent cell death, signifying apoptosis rather than
necrosis.
26
Here, treatment of HCT116N and HCT116O cells with [Rh(chrysi)(phen)(DPE-
Pt(NH
3
)
2
Cl)]
3+
in combination with caspase inhibitor results in a similar outcome. Cells
were treated with [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
(5 μM) or cisplatin (5 μM) in
combination with Z-VAD-FMK (35 μM) for 72h, and cell viability was determined by MTT
cytotoxicity assay (Figure 6).
A clear increase in cell viability upon addition of the caspase inhibitor is observed for both
cell lines treated with conjugate. In fact, caspase inhibition almost completely abolishes the
cytotoxic effects of the conjugate: the percentage of viable HCT116N cells increases from
74 ± 3.0% with [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
alone to 99 ± 1.6% upon addition of
Z-VAD-FMK, and the percentage of viable HCT116O cells is similarly enhanced from 81 ±
1.5% to 90 ± 2.7%. For both cell lines, these differences were determined to be statistically
significant by unpaired two-tailed
t
-test (
p
< 0.0001). These results, in combination with the
results of the MTT assay in combination with PARP inhibitor, signify that the cytotoxicity
of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
is caspase-dependent and PARP-independent.
[Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
induces an apoptotic mode of cell death in both
HCT116N and HCT116O cell lines.
The cisplatin-treated cells display similar results upon addition of Z-VAD-FMK: cell
viability increases 25 ± 2.9% and 13 ± 0.3% for HCT116N and HCT116O cells,
respectively, compared to treatment with cisplatin alone (
p
< 0.0001 by unpaired two-tailed
t
test). Exposure of cisplatin-treated cells to caspase inhibitor results in a markedly more
dramatic increase in cell viability compared to treatment with PARP inhibitor, suggesting
that while some cells may be undergoing necrotic death, the apoptotic pathway is likely the
major mechanism of cisplatin cytotoxicity.
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DISCUSSION
Synthesis of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
We have synthesized a new bimetallic Rh-Pt metalloinsertor derived from a recently
characterized family of complexes bearing axial Rh—O bonds.
24
Metalloinsertors
containing these ligands, which coordinate through a five-membered pyridylethanol ring,
have been shown to exhibit enhanced potency and cell-selectivity in MMR-deficient
cells.
22
,
24
Furthermore, these complexes can accommodate a wide variety of functional
groups incorporated into the
N,O
-coordinating ligand without sacrificing DNA binding
ability or biological activity, making this class of complexes an attractive scaffold for the
development of next-generation bifunctional metalloinsertor conjugates.
The metalloinsertor parent complex, [Rh(chrysi)(phen)(DPE)]
2+
(Figure 1), contains a non-
coordinating pyridine functionality within the pyridyl-ethanol ligand scaffold. This
extraneous pyridine serves as the site of coordination for cisplatin. Simple reflux of
commercially available cisplatin with the rhodium parent complex under acidic conditions
displaces one of the labile chloride ligands on the platinum center, affording [Rh(chrysi)
(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
(Figure 1) in a single step in reasonable yield. This conjugate,
then, contains a platinum center with only a single labilization site at the remaining chloride,
and is therefore expected to form “monofunctional” platinum adducts – that is, the platinum
will only coordinate a single nucleobase on the DNA, rather than binding two nearby
residues and forming the classical 1,2- or 1,3-intrastrand crosslinks characteristic of the
cisplatin parent complex.
Monofunctional platinum anticancer complexes, particularly those with the general structure
cis
-[Pt(NH
3
)
2
(L)Cl]
+
(where L is an N-heterocycle), have been heavily investigated by
Lippard and others.
38
41
Long considered to be clinically uninteresting owing to the lack of
activity of the first studied monofunctional compounds, [Pt(dien)Cl]
+
(dien =
diethylenetriamine) and [Pt(NH
3
)
3
Cl]
+,
42
44
interest in this class of complexes has been
renewed in recent years with the development of more active analogues, such as pyriplatin
(
cis
-[Pt(NH
3
)
2
(pyridine)Cl]
2+
)
45
and the highly potent phenanthriplatin (
cis
-
[Pt(NH
3
)
2
(phenanthridine)Cl](NO
3
)), which is being investigated as a new
chemotherapeutic agent.
46
,
47
These complexes have been shown to form monofunctional
adducts with single bases on DNA, usually at the
N
7 position of guanine.
38
,
45
Monofunctional adducts distort the DNA in a manner that is structurally distinct from that of
cisplatin and other doubly-coordinating
cis
-platinum (II) complexes, resulting in
considerably less bending and unwinding of the DNA.
48
51
These complexes thus exert their
anticancer activity via different biological mechanisms, providing orthogonality in the
treatment of cisplatin-resistant cancers.
48
In addition to the distinctive DNA binding
exhibited by
cis
-[Pt(NH
3
)
2
(L)Cl]
+
complexes, the presence of the bulky N-heterocycle
protects the metal center from deactivating protein thiols as well as recognition by
nucleotide excision repair proteins, which repair Pt-DNA adducts and lead to
resistance.
38
,
45
,
52
As a result, monofunctional, cationic platinum (II) complexes are a
growing class of platinum-based drugs that can be effective against cisplatin-resistant
cancers. Here, the synthesis and characterization of [Rh(chrysi)(phen)(DPE-Pt(NH
3
)
2
Cl)]
3+
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