Targeting DNA Mismatches with Rhodium Metalloinsertors
Kelsey M. Boyle
and
Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena CA
91125
Abstract
DNA has been exploited as a biological target of chemotherapeutics since the 1940s. Traditional
chemotherapeutics, such as cisplatin and DNA-alkylating agents, rely primarily on increased
uptake by rapidly proliferating cancer cells for therapeutic effects, but this strategy can result in
off-target toxicity in healthy tissue. Recently, research interests have shifted towards targeted
chemotherapeutics, in which a drug targets a specific biological signature of cancer, resulting in
selective toxicity towards cancerous cells. Here, we review a family of complexes, termed rhodium
metalloinsertors, that selectively target DNA base pair mismatches, a hallmark of mismatch-repair
(MMR) deficient cancers. These rhodium metalloinsertors, bind DNA mismatches with high
specificity and display high selectively in killing MMR-deficient versus MMR-proficient cells.
This cell selectivity is unique for small molecules that bind DNA. Current generations of rhodium
metalloinsertors have shown nanomolar potency along with high selectivity towards MMR-
deficient cells, and show promise as a foundation for a new family of chemotherapeutics for
MMR-deficient cancers.
Graphical Abstract Synopsis
Rhodium metalloinsertors selectively target DNA base pair mismatches, leading to the ejection of
the mismatched bases from the DNA
π
-stack. The selective binding of metalloinsertors to DNA
mismatches provides the basis for targeting mismatch-repair deficient cells as a strategy for new
chemotherapeutic design.
*
to whom correspondence should be addressed at jkbarton@caltech.edu.
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Keywords
Rhodium; Metalloinsertor; Mismatch Repair; DNA Probe
1. Introduction
DNA has proven to be a rich target for a large range of small-molecule therapeutic drugs.
The first DNA-targeting compounds with therapeutic properties were discovered in the
1940s [
1
,
2
]. Nitrogen mustards and antifolate drugs were found to cause tumor regression in
patients with non-Hodgkin's lymphoma and remission in children with lymphoblastic
leukemia, respectively [
2
,
3
]. It was found that the anticancer properties of these drugs arise
from their interactions with DNA; nitrogen mustards irreversibly alkylate DNA through an
aziridinium intermediate to form inter-strand crosslinks and antifolates block DNA synthesis
by inhibiting dihydrofolate reductase (DHFR), an enzyme necessary for the synthesis of
purine bases [
1
,
4
]. Since the discoveries of these therapeutics, the versatility of DNA as a
target has been significantly expanded. Therapeutics have been seen to bind covalently to
DNA (alkylating agents, platinum drugs), non-covalently interact with DNA (actinomycin
D, mitomycins, polyamides), to interfere with protein-DNA complexes (doxorubicin,
etoposide), and even target DNA secondary structures such as G-quadruplexes (intrafloxin,
in phase II clinical trials) [
5
–
8
]. These complexes and more DNA-targeting
chemotherapeutics have been reviewed recently [
5
,
9
].
In the development of novel metallodrugs, DNA is consistently one of the most exploited
targets. As seen in Figure 1, metal complexes can bind DNA through several different
routes, involving both covalent and non-covalent interactions [
10
,
11
]. In the covalent
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binding mode, the small molecule, for example cisplatin, binds directly to DNA to form a
covalent lesion, usually bonding with one or more bases and impeding DNA replication. In
contrast, non-covalent interactions rely on thermodynamic stabilization through
electrostatics, hydrogen bonding, hydrophobic interactions, and
π
-stacking interactions [
12
].
The majority of complexes that bind DNA non-covalently are either intercalators or groove
binders. In intercalation, an aromatic, heterocyclic ligand slips indiscriminately between two
adjacent base pairs. This process leads to a partial unwinding of the DNA, increasing the rise
of the helix. Groove binding is another common non-covalent binding motif of small
molecule therapeutics. In this binding mode, a small molecule that is generally crescent-
shaped tightly binds the minor groove of DNA. Unlike intercalators, which generally lack
sequence specificity, groove binders often target AT-rich regions. Moreover, sequence-
specific intercalators and groove binders have been prepared [
7
,
13
]. Once bound to DNA,
these non-covalent complexes primarily cause inhibition of proteins involved in DNA
transcription and synthesis, which can lead to cytotoxicity [
14
–
16
]. Somewhat recently, a
new non-covalent DNA binding mode, termed metalloinsertion, has been observed. In this
mode, a large aromatic, heterocyclic ligand inserts into DNA at a destabilized site and ejects
the destabilized bases from the helix, without causing an increase in base rise as is seen with
intercalators [
17
]. In contrast to intercalators, metalloinsertors are highly specific for
destabilized DNA mismatches, abasic sites, and single base bulges.
Though DNA-targeting metallodrugs are a significant field of study for many researchers,
few have had clinical success due to the general toxicity of heavy metals in the body [
18
–
20
]. The most noteworthy and well characterized metallodrug found to bind DNA is
cis
-
diamminedichloroplatinum(II) (cisplatin). Cisplatin, the first inorganic chemotherapeutic,
was discovered serendipitously by Barnett Rosenberg in 1965 while studying the effects of
electric fields on
E. coli
using a platinum electrode [
21
]. Today, cisplatin and its derivatives,
carboplatin and oxaliplatin, remain some of the most frequently used chemotherapeutics
with over 50% of all cancer regimens containing one of these drugs [
22
]. Once within a cell,
the chloride ligands of cisplatin are displaced by water [
23
]. This reactive intermediate binds
the N7 position of purine bases to form inter- and intra-strand DNA crosslinks, with the
biologically abundant adduct being 1,2-intrastrand crosslinks between two adjacent guanine
bases [
24
]. This adduct was structurally characterized using X-ray crystallography in 1995
[
25
]. The 2.6 Å resolution structure shows the bending of the DNA duplex by 40° towards
the major groove, accompanied by the widening of the minor groove. This lesion is
recognized intracellularly by DNA-binding proteins, eventually leading to the apoptotic
death of affected cells [
23
].
Despite its success in the clinic, cisplatin is not without its drawbacks. Patients treated with
cisplatin often experience severe, dose-limiting side effects such as nausea, vomiting,
nephrotoxicity, ototoxicity, and neurotoxicity [
26
]. These side effects occur because
cisplatin, like many chemotherapeutics, is not selective towards cancer cells—it binds DNA
inside healthy and cancerous tissues alike. Instead, cisplatin appears primarily to rely on
increased uptake by rapidly dividing cancer cells for effect [
5
]. Targeted therapy, in which a
specific biological signature of cancer drives preferential drug action on cancerous cells over
healthy cells, is a clear alternative to these non-specific chemotherapeutics. For example,
proteins that are upregulated or expressed exclusively in cancer cells may be exploited as
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cancer-selective targets [
27
]. For such protein targets, kinase inhibitors and monoclonal
antibodies have found clinical use in the treatment of a variety of cancers [
28
,
29
]. As an
example, cetuximab, a monoclonal antibody, targets and inhibits the epidermal growth factor
receptor (EGFR), which is upregulated in several cancers in order to maintain rapid
proliferation [
30
].
In addition to targeting specific proteins, it is also possible to target specific DNA lesions
associated with cancer, such as single base-pair mismatches. Over the last 30 years, our
laboratory has focused on targeting DNA mismatches using a unique family of complexes
called rhodium metalloinsertors. These complexes bind single base-pair mismatches
in vitro
and have been shown to be selectively cytotoxic towards mismatch repair deficient cell lines
[
17
,
31
]. This review is not an exhaustive examination of small molecules designed to target
DNA mismatches as this topic has been recently reviewed elsewhere [
32
]. Instead, this
article concentrates on the work of our own laboratory on this unique family of DNA-
binding complexes termed rhodium metalloinsertors.
2. Mismatch Repair Machinery
2.1 DNA Damage and Errors in Replication
The DNA within cells is constantly subject to damage by exogenous agents, such as UV
light and ionizing radiation, and endogenous modifications, such as depurination,
methylation, and errors in replication [
33
]. Some estimates suggest cells experience up to
10
5
such lesions each day [
34
]. This damage can lead to interruptions in cellular processes,
cell death, and mutations if uncorrected. High fidelity of DNA is essential, and therefore
cells have evolved complicated systems to repair many types of DNA damage, known
collectively as the DNA damage response. The DNA damage response consists of several
processes that identify or correct a broad range of damage, including base excision repair,
nucleotide excision repair, mismatch repair (MMR), and double strand break repair. These
processes have all been reviewed recently [
35
–
38
].
MMR machinery is primarily responsible for identifying and correcting replication errors in
the form of DNA base-pair mismatches and small insertions and deletions (indels). These
lesions result from failed proofreading of replication polymerases and polymerase slippage
during replication, respectively. The high fidelity of polymerases and their proofreading
exonuclease activities result in a low error rate of
∼
10
-7
mismatches per base pair per
replication, and this is improved upon by the MMR machinery, which increases fidelity an
additional 50-1000-fold [
39
]. Indels are generated more frequently, especially in repetitive
sequences [
35
,
40
].
2.2 Mechanisms of the MMR Machinery
The MMR machinery is responsible for identifying and correcting mismatches and indels in
newly synthesized DNA, as depicted in Figure 2. This process involves several major steps.
In the first step MutS
α
(heterodimer of MSH2 and MSH6) or MutS
β
(heterodimer of
MSH2 and MSH3) recognize and bind the mismatched region. MutS
α
, which contains 85%
of cellular MSH2, is responsible for recognizing and binding all base pair mismatches and
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1-2 base pair indels while Muts
β
can only efficiently repair indels [
35
,
41
]. Next, though the
mechanism is not well understood, MutS
α
undergoes a mismatch and ATP-dependent
conformational change that allows for the binding of MutL
α
, a heterodimer containing
MLH1 and PMS2. It is believed that MutL
α
, which has endonuclease activity when
activated by proliferating cell nuclear antigen (PCNA), identifies and nicks the nascent
strand of DNA, initiating excision of the DNA strand containing the replication error [
42
].
Several models exist that explain the removal of the mismatch in both the 5
′
-to-3
′
and 3
′
-
to-5
′
directions. Excision is followed by resynthesis of DNA and ligation. A detailed review
on current models in MMR has recently been published [
35
].
Deficiencies in either MutS
α
or MutL
α
have been associated with a loss of MMR
proficiency. MMR deficiencies result in a 50-1000 fold increase in mismatches within the
cell. When these mismatches are left unrepaired, they can propagate to form potentially
catastrophic mutations in future generations of cells. As such, deficiencies in MMR
machinery are associated with many forms of cancer, including nearly 80% of hereditary
non-polyposis colon cancers and 15-20% of all solid tumors [
43
,
44
]. Additionally, these
cancers often show resistance to traditional chemotherapeutics, such as cisplatin and
alkylating agents, making them excellent candidates for targeted therapy [
45
].
3. Binding of Rhodium Metalloinsertors to DNA Mismatches
3.1 Designing a mismatch-targeting molecule
Our laboratory has explored complexes that can non-covalently target specific DNA
sequences (such as 5
′
-TGCA-3
′
and 5
′
-py-py-pu-3
′
sites) as well as the non-conventional
A- and Z- forms of DNA, but these targets are not implicated in disease and thus lack
therapeutic potential [
13
,
46
–
48
]. DNA mismatches, however, are generic DNA targets that
are involved in many types of cancer, as described above. Due to imperfect hydrogen
bonding and
π
-stacking, DNA base pair mismatches are thermodynamically destabilized
compared to well-matched DNA [
49
]. This slight destabilization has been successfully
targeted through the use of rhodium metalloinsertors, which contain the sterically expansive
5,6-chrysenequinone diimine (chrysi) ligand.
The chrysi ligand was designed to be larger than traditional intercalating ligands and more
akin in size to a well-matched base pair, making it too bulky to simply intercalate into DNA
(Figure 3) [
16
]. Instead, chrysi interacts with DNA through insertion at a destabilized site.
Insertion, which was originally proposed by L. S. Lerman in 1961, is a DNA binding mode
in which a DNA base pair is separated and ejected from the
π
-stack by the inserting
molecule [
50
]. Rhodium(III) was chosen to be a substitutionally inert metal anchor for the
chrysi ligand due to its photophysical properties; the rhodium complexes promote DNA
strand scission in related metallointercalators with photoexcitation [
51
]. The rhodium center
also anchors two ancillary ligands, which add bulk to the complexes and limit how the chrysi
ligand can interact with DNA, largely preventing indiscriminant intercalation [
52
]. These
ancillary ligands can also be selected to tune DNA binding and cellular uptake properties, as
discussed below.
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3.2 Targeting DNA mismatches with Rhodium Metalloinsertors
The first generation metalloinsertor, [Rh(bpy)
2
(chrysi)]
3+
(bpy = 2,2
′
-bipyridine), is easily
synthesized via a base-catalyzed condensation reaction between [Rh(bpy)
2
(NH
3
)
2
]
3+
and
5,6-chrysene quinone [
53
]. As predicted, [Rh(bpy)
2
(chrysi)]
3+
can selectively bind DNA
base pair mismatches with nanomolar affinities and photocleave the DNA backbone adjacent
to the mismatch when irradiated with UV-light [
17
]. This process is enantioselective,
meaning only the right handed Δ-enantiomer can bind right handed, B-form DNA. A series
of binding affinity assays were employed to determine the specificity of
[Rh(bpy)
2
(chrysi)]
3+
. For instance, when incubated with a 2725 base pair linearized plasmid
containing a single CC mismatch, [Rh(bpy)
2
(chrysi)]
3+
selectively binds and photocleaves
the DNA solely at the mismatched site [
54
]. No photocleavage is observed with the
analogous well-matched plasmid. [Rh(bpy)
2
(chrysi)]
3+
was also incubated with DNA
segments containing all possible DNA mismatches and multiple different sequence contexts
in which the base pairs flanking the mismatch were varied [
55
]. Through these experiments,
it was determined that [Rh(bpy)
2
(chrysi)]
3+
has >1000-fold preference for targeting
mismatched sites over well-matched sites. Additionally, the complex binds and cleaves 80%
of all DNA mismatches upon irradiation, irrespective of sequence context. Not surprisingly,
the binding affinity of [Rh(bpy)
2
(chrysi)]
3+
towards a mismatch correlates strongly with the
thermodynamic destabilization of the mismatch; highly destabilized mismatches (such as
CC, CA, and CT mismatches) are easily recognized by [Rh(bpy)
2
(chrysi)]
3+
whereas more
stabilized mismatches, specifically mismatches containing guanine, are not preferentially
bound by the complex.
A crystal structure at 1.1 Å resolution of [Rh(bpy)
2
(chrysi)]
3+
with a 12-mer oligonucleotide
containing two AC mismatches further elucidated the binding mode of the complex to be
metalloinsertion [
56
]. Unlike classical metallointercalators, the metalloinsertor binds DNA
via the minor groove and results in little distortion to the DNA backbone. Instead, the DNA
accommodates the inserting ligand through the ejection of the mismatched bases out of the
π
-stack and into the major and minor grooves. This binding mode was verified with an
additional crystal structure of the complex bound to an AA mismatch (Figure 4), as well as a
solution NMR structure of the complex with DNA containing a CC mismatch [
57
,
58
]. This
structure provides additional insight into why G-containing mismatches are not detected by
metalloinsertors; these highly stable mismatches are not easily ejected from the base-stack,
so chrysi cannot displace mismatches at these sites.
4. Biological Activity of Rhodium Metalloinsertors
4.1 Targeting MMR-deficiencies with Rhodium Metalloinsertors
The therapeutic potential of rhodium metalloinsertors was explored after experiments
showed their ability to bind DNA base pair mismatches selectively. It was hypothesized that
metalloinsertors would have increased toxicity towards cells that contain an increased
number of DNA mismatches, as is present in MMR-deficient cells. To test this hypothesis,
an enzyme-linked immunosorbent assay (ELISA) for cellular proliferation was performed
with two colorectal cancer cell lines, the HCT116O cell line, which is MMR-deficient, and
the MMR-proficient HCT116N cell line. These cell lines, which originate from the MLH1
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deficient HCT116 parent cell line, are isogenically matched; the HCT116N cell line is
transfected with human chromosome 3, which encodes for a functioning MLH1 gene, and
the HCT116O cell line is transfected with human chromosome 2, leaving it MMR-deficient
[
59
]. [Rh(bpy)
2
(chrysi)]
3+
was found to have increased potency in the MMR-deficient cell
line [
60
]. These complexes have also been seen to bind abasic sites and single-base bulges in
DNA [
61
,
62
].
As with the DNA binding studies, only the Δ enantiomer was biologically active, suggesting
the compounds do not decompose or racemize within the cell. This unique activity, the
ability to selectively kill MMR-deficient cells over their MMR-proficient counterparts, has
been found to be common to many rhodium metalloinsertor [
31
,
63
,
64
]. Importantly, while
general to metalloinsertors, these are the only complexes known to exhibit this type of
selectivity, with common chemotherapeutics such as cisplatin and the DNA-alkylating agent
N-methyl-N
′
-nitro-N-nitrosoguanidine (MNNG) showing the opposite trend (Figure 5) [
45
].
These results have been additionally verified in a MSH2-deficient cell line, showing that the
characteristic activity of these complexes towards MMR-deficient cell lines is dependent on
the MMR-deficient phenotype rather than a unique feature of the HCT116 cell lines [
65
].
That the complexes show cell-selective inhibitory activity is somewhat surprising, however,
considering that metalloinsertors interact only non-covalently with DNA and even MMR-
deficient cells contain relatively few mismatches. Like other non-covalent DNA intercalators
and groove binders, it is possible that rhodium metalloinsertors bind DNA mismatches and
disrupt transcription or replication processes, leading to selective cytotoxicity in MMR-
deficient cells. Ongoing research in our lab aims to better understand the activity of rhodium
metalloinsertors and the mechanisms that lead to this selective cell death.
4.2 DNA-binding affinity and subcellular localization of metalloinsertors
As discussed previously, the design of the inserting chrysi ligand was central to obtaining
mismatch specificity. It is important to note, however, that the design of the ancillary ligands
has proven also to be important. The metalloinsertion binding mode places the ancillary
ligands of the rhodium complexes in close proximity to the DNA bases and backbone. For
this reason, several structure-function studies have been performed to determine the effect of
the ancillary ligands on biological activity. In one study, the ancillary ligands were varied in
size from small ammine groups to bulky 4,7-diphenyl-phenanthroline (DIP) groups [
31
].
The binding affinities to mismatched DNA spanned over 3 orders of magnitude and
correlated well to biological activity, with higher affinity mismatch-binding complexes being
more selective than their low-affinity counterparts. Again, none of the complexes showed
selective inhibition of the MMR-proficient cells.
Differences in binding affinity, however, are not the sole predictor of biological activity. The
activities of two structurally similar metalloinsertors, [Rh(DPAE)
2
(chrysi)]
3+
and
[Rh(PrDPA)
2
(chrysi)]
3+
(DPAE = 2-(di(pyridin-2-yl)amino)ethanol, PrDPA =N-propyl-N-
(pyridin-2-yl)pyridin-2-amine), were examined to explore in more detail the importance of
uptake and subcellular localization (Figure 6) [
66
]. These two complexes have similar
mismatch binding affinities and differ only in the presence of either ethanol or propyl
modified HDPA (2,2
′
-dipyridylamine) ligands. The addition of these ligands does lead to a
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difference in lipophilicity, however, as measured by the partition constant. As expected, the
more lipophilic complex, [Rh(PrDPA)
2
(chrysi)]
3+
, showed significantly higher cellular
uptake than [Rh(DPAE)
2
(chrysi)]
3+
, making its non-selective biological activity initially
surprising. However, upon subcellular fractioning and analysis using inductively coupled
plasma spectrometry (ICP-MS), it became clear that the subcellular localization of these two
complexes provided an explanation for their activities. [Rh(PrDPA)
2
(chrysi)]
3+
showed a 10-
fold increase in mitochondrial uptake over [Rh(DPAE)
2
(chrysi)]
3+
. Although more
[Rh(PrDPA)
2
(chrysi)]
3+
was also found in the nucleus of the cells, the percentage of total Rh
found within the nucleus is higher for [Rh(DPAE)
2
(chrysi)]
3+
. These results suggest that
localization of complexes to the mitochondria eradicates their biological selectivity for
MMR-deficient cells and leads to MMR-independent death of both cell lines. Interestingly,
these findings also support the hypothesis that metalloinsertors achieve their cell selectivity
through binding
nuclear
DNA mismatches, not mitochondrial DNA.
The effects of subcellular localization were further examined with a larger family of
complexes that differed primarily in lipophilicity [
63
]. With the exception of
[Rh(DIP)
2
(chrysi)]
3+
, all complexes display similar binding affinities to mismatched DNA
in the 10
6
to 10
7
M
-1
range. Once again, it was found that biological selectivity correlated
not with overall cellular uptake or nuclear localization, but instead with mitochondrial
localization. As expected, the more lipophilic, greasy cations showed the highest
mitochondrial localization, which correlated with elimination of biological selectivity [
67
].
The more hydrophilic complexes had significantly lower localization to the mitochondria,
which correlated with higher cell selectivity. These studies highlighted the importance not
only of cellular uptake but also considerations of subcellular localization. These studies
furthermore suggested that off-target effects, such as seen with mitochondrial targeting, are
detrimental to the biological function of metalloinsertors.
4.3 Conjugation of Metalloinsertors to cytotoxic agents
In an effort to increase the potency of metalloinsertors and obtain a covalent complex with
DNA, our laboratory synthesized several new complexes that conjugate a selective rhodium
metalloinsertor to a potent cytotoxic agent [
68
–
70
]. Three conjugates were designed that
combine a reactive platinum moiety with a rhodium metalloinsertor (Figure 7) [
69
,
71
,
72
].
The conjugates were constructed to work through three different mechanisms. RhPt1
permanently tethers a Pt moiety to the metalloinsertor through a long flexible chain, which
allows for concurrent binding of both the Pt center and the metalloinsertor. In theory, the
metalloinsertor would bind a mismatch and guide the Pt moiety to a nearby GG on the same
DNA strand. In experiments monitoring binding to oligonucleotides containing mismatches,
increased platination of DNA containing the mismatch was observed, but significant
platination of well-matched DNA was still present. In a different approach, RhPt2 was
designed to contain an oxaliplatin moiety that can dissociate from the metalloinsertor.
Ideally, after the metalloinsertor binds mismatched DNA, the oxaliplatin moiety would
dissociate and bind the same DNA strand nearby. Once again, increased platination was
observed for mismatched DNA, but the complex was not biologically selective. Finally,
RhPt3 contains a short tether to a monofunctional Pt moiety. In contrast to RhPt1, this short
tether would force Pt to bind DNA at the mismatched site following metalloinsertion.
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Although significantly more potent than cisplatin, this complex was also not selective
towards MMR-deficient cells.
In all three experiments, the selective metalloinsertor was designed to bind a mismatch and
therefore preferentially guide the covalent DNA-binding platinum moiety to mismatched
DNA. However, these complexes do not exhibit selective cytotoxicity towards MMR-
deficient cells. Even RhPt2, which showed increased platination of mismatched DNA
in
vitro
, is not specific for MMR-deficient cells. It is likely that the differences between
traditional rhodium metalloinsertors and RhPt conjugates are caused by the mode of cell
death initiated by these complexes. RhPt conjugates undergo apoptotic cell death, similar to
their parent complexes cisplatin and oxaliplatin. This result is in stark contrast to the
distinctly necrotic cell death caused by traditional rhodium metalloinsertors. These
observations suggest that initiation of necrotic cell death may be essential to the selective
activity of metalloinsertors. It is possible that RhPt conjugates promote both modes of cell
death, but the apoptotic pathway overrides the necrotic pathway, leading to biological
activity reminiscent of traditional platinum drugs.
5. Current Design of Rhodium Metalloinsertors
Recently, a new family of potent rhodium metalloinsertors based on [Rh(DPE)(chrysi)
(phen)]
2+
(DPE=1,1-di(pyridine-2-yl)ethan-1-ol, phen=1,10-phenanthroline) has been
examined (Figure 8) [
64
]. Unlike previous generations of metalloinsertors, these
complexescontain an unusual Rh-O bond through the coordination of their pyridyl-ethanol
ligands. These Rh-O containing metalloinsertors are more potent than cisplatin and display
optimal differential cellular activity in the nanomolar range, as much as two orders of
magnitude more potent than earlier generations of complexes. Surprisingly, this scaffold is
robust to many substitutions of the oxygen-containing ligand: replacing the dangling pyridyl
group of DPE with a small methyl group, a phenyl group, or a greasy hexyl group all lead to
improved, nanomolar cytotoxicity in MTT assays. Furthermore, and surprisingly, both the Δ
and
Λ
enantiomers of these new complexes bind DNA with similar affinity
in vitro
and both
show differential cell-selective activity in MTT assays.
Remarkably, the increased potency and selectivity of [Rh(DPE)(chrysi)(phen)]
2+
and its
derivatives is not a result of increased DNA binding or localization. For example, in
comparison to the complex [Rh(phzi)(NH3)4]
3+
, a selective metalloinsertor that utilizes the
expansive benzo[
α
]phenazine quinone diimine (phzi) ligand, [Rh(DPE)(chrysi)(phen)]
2+
possesses an order of magnitude lower mismatch binding affinity, similar cellular uptake and
mitochondrial concentrations, and slightly lower nuclear concentration. Despite these
differences, [Rh(DPE)(chrysi)(phen)]
2+
shows comparable selectivity towards MMR-
deficient cells as the tetrammine complex but is nearly 4-fold more potent than [Rh(phzi)
(NH3)4]
3+
. Initially, it seemed possible that the increased potency and racemic binding
could be due to a labile Rh-O bond, leading to covalent DNA binding and racemization
within the cell. However, these possibilities were eliminated using several
in vitro
tests of
stability. It appears that the enantiomeric activity of these complexes is authentic, with both
Δ and
Λ
enantiomers being able to kill MMR-deficient cells selectively without
racemization.
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One apparent difference between the Rh-O containing metalloinsertors and previous
generations are dramatic changes in the pKa of the chrysi imine protons. The pKa of the first
generation complex, [Rh(bpy)
2
(chrysi)]
3+
, is 5.6 ± 0.2; thus the complex is deprotonated at
cellular pH. The deprotonation of the chrysi ligand relieves steric clashes between its imine
proton and aromatic ring system protons, allowing the chrysi ligand to lay planar (Figure 8).
This planarity can be seen in the crystal structure of [Rh(bpy)
2
(chrysi)]
3+
bound to a DNA
mismatch. The Rh-O containing metalloinsertors have significantly higher pKa values of
8.3-8.9, meaning the chrysi ligand cannot deprotonate at cellular pH. To relieve steric
clashing, the chrysi ligand must instead buckle relative to [Rh(bpy)
2
(chrysi)]
3+
(Figure 8).
This buckling can be seen in the crystal structure of [Rh(DPE)(chrysi)(phen)]
2+
. While we
still believe the binding mode of these complexes to be metalloinsertion, it appears that this
significant structural change in the inserting ligand of these complexes must create a slightly
different DNA lesion than [Rh(bpy)
2
(chrysi)]
3+
. The new lesion or lesions must
accommodate both the Δ and
Λ
enantiomers. Within the cell, this lesion may be more easily
recognized by proteins activating necrotic cell death than lesions caused by previous
metalloinsertors, leading to the increased potency and selectivity of these complexes. Clearly
much needs to be done to elucidate the basis of the high potency and cell selectivity of these
new complexes, both from a structural standpoint and with regard to understanding the
biological fate of the complexes.
6. Future Outlook
Rhodium metalloinsertors constitute a unique family of transition metal complexes that
selectively bind DNA base pair mismatches and preferentially inhibit proliferation and
survival of MMR-deficient cells. Over the years, the design of these complexes has
improved significantly, traversing from micromolar toxicities now into the nanomolar range.
Thus a unique family of rhodium metalloinsertors with nanomolar potencies and high
selectivity for MMR-deficient cell lines has now been characterized, bringing these
complexes into the realm of therapeutic interest [
64
].
Despite improvements to the design of these complexes, however, relatively little is still
understood with respect to their mechanism of action within the cell. The complexes possess
similar, if not identical binding properties and localization patterns to previous generations
of metalloinsertors, yet they are significantly more potent and selective. Additionally, from a
biological standpoint, it seems incredible that the modest increase in DNA lesions associated
with MMR-deficiencies can be so selectively targeted by these complexes. Clearly future
studies must be carried out to better understand the selective activity of these complexes,
specifically to identify biological pathways or specific proteins that may recognize the
metalloinsertor-DNA lesion.
Based on the metalloinsertive binding mode of [Rh(bpy)
2
(chrysi)]
3+
, elucidated through
solid and solution state structures, as well as
in vitro
and
in cellulo
work performed on the
extensive family of metalloinsertors, we hypothesize that these complexes bind DNA
mismatches within MMR-deficient cells, and that this Rh-DNA lesion is recognized by
proteins and cellular machinery that eventually lead the cell to necrosis. Unlike cisplatin,
these complexes do not appear to form covalent adducts with DNA. Instead, it is possible
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that the non-covalent binding of metalloinsertors inhibits proteins involved in DNA
processes such as transcription or replication, similar to other non-covalent DNA groove
binders and intercalators. In such a case, their activity may only be evident in MMR-
deficient cells containing increased concentrations of mismatches, leading to their observed
selectivity. It is possible that the lesion formed by the more potent and selective complexes
of the current generation, that can accommodate the Δ and
Λ
enantiomer, may also be more
recognizable in the cell, or processed more efficiently to produce necrosis. Future work on
metalloinsertors will emphasize characterizing the structures of metalloinsertors bound to
their target mismatches, understanding the biological mechanisms of metalloinsertors,
identifying proteins and pathways involved in their processing, and developing future
generations of metalloinsertors with improved potency or selectivity for use as a foundation
for a new family of chemotherapeutics.
Indeed what has been clear from these studies at the outset is that transition metal chemistry
offers a rich variety of means to target DNA sites along with novel methods to characterize
their fates inside cells. Cisplatin, despite its simplicity in structure and relative lack of
specificity in cellular targeting, has proven to be a powerfully important therapeutic. We
expect that greater specificity in cellular targeting along with greater specificity in DNA
targeting can only aid us in the development of new strategies upon which to build and
potentially new potent and selective metal compounds as cancer therapeutics.
Acknowledgments
We are grateful to our coworkers and collaborators in elucidating this chemistry and to the NIH for their support of
this research (GM33309). We also thank the United States Department of Defense for an NDSEG research
fellowship to K.M.B.
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Abbreviations
MMR
mismatch repair
DHFR
dihydrofolate reductase
EGFR
epidermal growth factor receptor
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PCNA
proliferating cell nuclear antigen
chrysi
5,6-chrysene quinone diimine
bpy
2,2
′
-bipyridine
ELISA
enzyme-linked immunosorbent assay
MNNG
N-methyl-N
′
-nitro-N-nitrosoguanidine
DIP
4,7-diphenyl-phenanthroline
DPAE
2-(di(pyridine-2-yl)amino)ethanol
PrDPA
N-propyl-N(pyridine-2-yl)pyridine-2-amine
HDPA
2,2
′
-dipyridylamine
ICP-MS
inductively coupled plasma-mass spectrometry
DPE
1,1-di(pyridine-2-yl)ethan-1-ol
phen
1,10-phenanthroline
phzi
benzo[a]phenazine quinone diimine
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Highlights
•
Cancers associated with MMR deficiencies are often resistant to
chemotherapeutics
•
Rhodium metalloinsertors selectively bind DNA base pair mismatches
•
Rhodium metalloinsertors are preferentially cytotoxic to MMR
deficient cell lines
•
Recent rhodium metalloinsertors have improved nanomolar potency
and selectivity
•
Improved metalloinsertors show therapeutic promise for MMR
deficient cancers
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Figure 1.
Four common binding modes of small molecules to DNA: (a) covalent binding, (b)
intercalation, (c) groove binding, and (d) insertion. Structural data from PDB files 1AIO,
454D, 2LWH, and 3GSK.
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Figure 2.
Propagation of an AA mismatch through multiple replications, resulting in a TA mutation.
The first iteration of replication results in an AA mismatch, shown in red. The mismatch can
be processed and repaired by the MMR machinery shown at the bottom. If unprocessed,
upon a second iteration of replication the mismatch will result in a mutation, shown in red.
Newly synthesized DNA from the first iteration and second iterations are shown in blue and
green, respectively.
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Figure 3.
Comparison of the well matched GC and AT mismatches to the non-specific intercalating
ligand, phi, and the mismatch-specific inserting ligand, chrysi. Representative rhodium
complexes, [Rh(bpy)
2
(phi)]
3+
(blue) and [Rh(bpy)
2
(chrysi)]
3+
(red) are shown. Chrysi is 0.5
Å wider than well-matched base pairs and 2.1 Å larger than phi.
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