A Family of Rhodium Complexes with Selective Toxicity towards
Mismatch Repair-Deficient Cancers
Kelsey M. Boyle
and
Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena
CA, 91125
Abstract
Rhodium metalloinsertors are a unique set of metal complexes that bind specifically to DNA base
pair mismatches
in vitro
and kill mismatch repair (MMR)-deficient cells at lower concentrations
than their MMR-proficient counterparts. A family of metalloinsertors containing rhodium-oxygen
ligand coordination, termed “
Rh–O
” metalloinsertors, has been prepared and shown to have a
significant increase in both overall potency and selectivity towards MMR-deficient cells regardless
of structural changes in the ancillary ligands. Here we describe DNA-binding and cellular studies
with the second generation of
Rh–O
metalloinsertors in which an ancillary ligand is varied in both
steric bulk and lipophilicity. These complexes, of the form [Rh(L)(chrysi)(PPO)]
2+
, all include the
O-containing PPO ligand (PPO = 2-(pyridine-2-yl)propan-2-ol) and the aromatic inserting ligand
chrysi (5,6-chrysene quinone diimine) but differ in the identity of their ancillary ligand L, where L
is a phenanthroline or bipyridyl derivative. The
Rh–O
metalloinsertors in this family all show
micromolar binding affinities for a 29-mer DNA hairpin containing a single CC mismatch. The
complexes display comparable lipophilic tendencies and pK
a
values of 8.1–9.1 for dissociation of
an imine proton on the chrysi ligand. In cellular proliferation and cytotoxicity assays with MMR-
deficient cells (HCT116O) and MMR-proficient cells (HCT116N), the complexes containing the
phenanthroline-derived ligands show highly selective cytotoxic preference for the MMR-deficient
cells at nanomolar concentrations. Using mass spectral analyses, it is shown that the complexes are
taken into cells through a passive mechanism and exhibit low accumulation in mitochondria, an
off-target organelle that, when targeted by parent metalloinsertors, can lead to non-selective
cytotoxicity. Overall, these
Rh–O
metalloinsertors have distinct and improved behavior compared
to previous generations of parent metalloinsertors, making them ideal candidates for further
therapeutic assessment.
Graphical Abstarct
*
to whom correspondence should be addressed at jkbarton@caltech.edu.
Associated Content
Some experimental methods, representative binding affinity determination, pH titrations, enantiomeric characterization, further DNA
binding characterization, and cytotoxicity of the two enantiomers as well as cellular uptake data. This material is available free of
charge via the Internet at
http://pubs.acs.org
.
Notes: The authors declare no competing financial interest.
HHS Public Access
Author manuscript
J Am Chem Soc
. Author manuscript; available in PMC 2018 May 23.
Published in final edited form as:
J Am Chem Soc
. 2018 April 25; 140(16): 5612–5624. doi:10.1021/jacs.8b02271.
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INTRODUCTION
Over the past 70 years, DNA and its associated metabolic processes have proven to be
fruitful targets for the design of new therapeutic agents.
1
Many of the most common FDA-
approved chemotherapeutics work by binding DNA, such as the DNA-crosslinking agent
cisplatin and the DNA-intercalating agent doxorubicin.
2
–
5
Despite the prevalence of these
drugs in the clinic, there are many drawbacks to their design and mechanisms of action. In
many cases, the drugs target a generic DNA structure that is common to both healthy and
cancerous cells. The incidental targeting of healthy tissue can result in dramatic and often
dose-limiting side effects, such as emesis and nephrotoxicity.
6
To circumvent these off-target
effects, it is essential to identify new therapeutic targets that are almost exclusively found
within cancerous tissues and cells.
In our research, we focus on one such target: DNA base pair mismatches. Mismatches occur
regularly in cells due to polymerase errors or interaction with exogenous compounds.
7
In
healthy cells, these errors are corrected by the mismatch repair (MMR) machinery of the
cell. However, in many solid tumors or tumors of Lynch syndrome patients, mutations in
MMR proteins severely down-regulate or completely inactivate repair.
8
,
9
As a result, these
cancers contain a relative abundance of DNA base pair mismatches compared to healthy
cells, making mismatches a potential biomarker for selective cancer therapy.
Mismatched base pairs have been targeted through the design of metal complexes, called
rhodium metalloinsertors, which selectively and non-covalently bind these lesions.
10
Rhodium metalloinsertors contain a sterically expansive aromatic chrysi (5,6-
chrysenequinone diimine) ligand that is capable of
π
-stacking with DNA bases. Due to
steric bulk, however, the chrysi ligand is unable to easily intercalate into well-matched DNA,
and instead primarily interacts with DNA at thermodynamically destabilized sites, such as
mismatches or abasic sites.
11
The ability of a prototypical metalloinsertor,
[Rh(bpy)
2
(chrysi)]
3+
(bpy = 2,2
′
-bipyridine), to selectively bind DNA mismatches has been
verified using both
in vitro
binding assays and crystallographic studies.
12
–
15
Crystallographic and NMR studies show that this complex binds DNA mismatches
via
metalloinsertion, a non-covalent binding mode in which the complex inserts into DNA at the
mismatched site from the minor groove, ejects the mismatched DNA bases, and
π
-stacks
with the flanking well-matched base pairs.
14
This mismatch-targeting ability has also been
seen in human cell culture experiments, with metalloinsertors exhibiting enhanced
cytotoxicity in MMR-deficient cell lines relative to their MMR-proficient counterparts.
15
,
16
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This result is in stark contrast to most DNA-targeting therapeutics, such as the
aforementioned cisplatin and doxorubicin, which are selective towards MMR-
proficient
cell
lines over MMR-deficient cell lines, leading to the development of resistance in MMR-
deficient tumors following treatment.
17
,
18
Several generations of metalloinsertors have been synthesized since [Rh(bpy)
2
(chrysi)]
3+
,
which has led to the recent discovery of a potent and selective family of rhodium
metalloinsertors containing a pyridyl-alcohol ligand and unique Rh–O ligand coordination
(Figure 1).
19
This Rh–O ligand coordination is structurally distinct from earlier generations
of parent metalloinsertors, which contained solely Rh–N coordination.
20
Furthermore, these
Rh–O
metalloinsertors were found to have improved potency and selectivity towards MMR-
deficient cancer cells over MMR-proficient cancer cells. Surprisingly, this high potency and
cell selectivity was seen across a variety of metalloinsertors containing O-coordinated
ligands that differed significantly in size and structure (spanning methyl, pyridyl, phenyl,
and hexyl functionalization), suggesting the biological activities of
Rh–O
metalloinsertors
are not perturbed by ligand substitution off of the O-containing site.
Here, a family of rhodium metalloinsertors was designed and synthesized as variations of the
Rh–O
metalloinsertor [Rh(phen)(chrysi)(PPO)]
2+
(phen = 1,10-phenanthroline). These
complexes, of the form [Rh(L)(chrysi)(PPO)]
2+
, all include the O-containing PPO ligand but
differ in the identity of their ancillary ligand, L, where L= bpy, HDPA (2,2
′
-
dipyridylamine), 4,7-DMP (4,7-dimethyl-1,10-phenanthroline), 5,6-DMP (5,6-
dimethyl-1,10-phenanthroline), and DIP (4,7-diphenyl-1,10-phenanthroline) (Figure 2). The
ancillary ligand substitution alters the steric bulk and lipophilicity of these complexes, which
can ultimately affect DNA-binding properties and biological activity.
20
,
21
Each complex
described, even the most lipophilic and sterically bulky, shows biological selectivity towards
MMR-deficient cell lines, further demonstrating that the Rh–O ligand framework is
amenable to a wide array of functionalization. To better understand the trends in biological
activity of these complexes, each metalloinsertor was examined for binding affinity to
mismatched DNA, pK
a
, lipophilicity, whole cell uptake, and subcellular localization into the
nucleus and mitochondria. The results indicate that minimizing uptake of the complexes into
the mitochondria may be a key factor in ensuring high biological selectivity and support that
these
Rh–O
complexes exhibit distinct differences in metalloinsertor-DNA binding and cell
activation compared to parent metalloinsertors.
EXPERIMENTAL METHODS
Materials
Commercially available chemicals were used as received. All reagents and Sephadex ion-
exchange resin were obtained from Sigma-Aldrich with the following exceptions. RhCl
3
was
purchased from Pressure Chemical, Inc. Dowex ion-exchange beads were purchased from
Acros Organics. Analytical standards for Rb and transition metals were purchased from
Analytical West and Ultra Scientific, respectively. MTT and ELISA assay kits were obtained
from Roche. Pierce BCA assay kit and NP40 were purchased from Thermo Scientific. Sep-
pak C18 solid-phase extraction (SPE) cartridges were purchased from Waters Chemical Co.
Cell culture media and supplements were purchased from Invitrogen. Tissue culture flasks
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and plates were obtained from Corning.
32
P labeled ATP was purchased from Perkin Elmer.
UreaGel supplies were purchased from National Diagnostics. Microbiospin columns were
purchased from BioRad.
Synthesis and Characterization of Metal Complexes
[Rh(phen)(chrysi)(PPO)]Cl
2
and [Rh(bpy)
2
(chrysi)]Cl
3
were synthesized following the
published protocols.
19
,
22
New metal complexes were synthesized in a similar manner to
published procedures.
19
,
20
,
23
A description of the general synthetic procedures are below.
Complete synthetic details for each complex, including specific amounts (masses, volumes,
and ratios) as well as slight deviations from the synthetic scheme below, can be found in the
SI.
Synthesis of [Rh(L)Cl
4
][K or H
3
O] complexes
For L = bpy, 4,7-DMP, 5,6-DMP, and DIP: RhCl
3
•3H
2
O (1 equiv.) and KCl (1 equiv.) were
refluxed in methanol for 2 h at 98 °C. Ligand (L, 1 equiv.) was added in a minimum volume
of methanol and refluxed for 4 h, during which the deep red solution turned to golden-brown
precipitate. The solution was filtered over a medium fritted filter, rinsed with methanol, and
dried under vacuum to produce [Rh(L)Cl
4
]K. Crude yield: 84% (bpy), 86% (4,7-DMP), 91%
(5,6-DMP), 95% (DIP). For L = HDPA: RhCl
3
•3H
2
O (1 equiv.) was refluxed in
concentrated HCl (38% w/v) for 3 h at 98 °C. Ligand (L, 2 equiv.) was added in a minimum
volume of HCl, followed immediately by boiling water. The solution was refluxed for 16 h,
then cooled to 4 °C. The golden precipitate was filtered over a Buchner funnel and dried
under vacuum to produce [Rh(L)Cl
4
][H
3
O]. Crude yield: 100% (HDPA).
Synthesis of [Rh(L)(NH
3
)
4
][OTf]
3
[Rh(L)Cl
4
][K or H
3
O] (1 equiv.) was added to an oven-dried 25 mL Schlenk flask and
degassed under argon. Neat triflic acid (HOTf, 10 g, excess) was added to the flask under
positive argon pressure, producing a deep red solution. The flask was purged to remove
newly formed HCl gas and stirred for 16 h. The solution was then added dropwise to cold,
stirring ether at −78 °C to produce a yellow-brown precipitate. The precipitate was filtered
over a medium fritted filter and rinsed with additional cold ether. The product, [Rh(L)
(OTf)
4
][K or H
3
O], was combined with NH
4
OH (28% w/v) and stirred at 40 °C for 1 h. The
solvent was removed under vacuum and the product was suspended in minimal ethanol,
precipitated with ether, filtered over a medium fritted filter, and dried further under vacuum
to produce [Rh(L)(NH
3
)
4
][OTf]
3
. Crude yields of 42% (bpy), 10% (HDPA), 15% (4,7-
DMP), 77% (5,6-DMP), 72% (DIP).
Synthesis of [Rh(L)(chrysi)(NH
3
)
2
][OTf]
3
[Rh(L)(NH
3
)
4
][OTf]
3
(1 equiv.) was combined with 5,6-chrysene-quinone (1 equiv.) and a
mixture of acetonitrile, water, and NaOH, and stirred for 1–12 h at ambient temperature. The
solution changed from bright orange (the color of free ligand) to red-brown (for L = bpy,
HDPA, 5,6-DMP, and DIP) or green-brown (for L=4,7-DMP) with no precipitate. The
reaction was quenched with HCl, producing a deep red solution, and the solvent was
removed under vacuum. The products from L=bpy, HDPA, and DIP were purified using a
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C18 SepPak, pre-equilibrated with 0.1% TFA (aq, TFA = trifluoroacetic acid) and eluted
with 1:3 MeCN:0.1% TFA (aq). The products from L=4,7-DMP and 5,6-DMP were purified
by HPLC using a reverse phase C18 column with gradient elution from 15:85 MeCN:0.1%
TFA (aq) to 95:5 MeCN:0.1% TFA (aq) over 30 min. Products were in the form of [Rh(L)
(chrysi)(NH
3
)
2
][TFA]
3
. Crude yields of 33% (bpy), 51% (HDPA), 46% (4,7-DMP), 62%
(5,6-DMP), 100% (DIP).
Synthesis of [Rh(L)(chrysi)(PPO)]Cl
2
[Rh(L)(chrysi)(NH
3
)
2
][TFA]
3
(1 equiv.) was combined with PPO in a mixture of ethanol
and water and refluxed 16 h (for L = bpy, 4,7-DMP, 5,6-DMP, and DIP) or 7 days (for
L=HDPA). The solvent was removed under vacuum and the product was purified by HPLC
using the method described above for L = bpy, HDPA, 4,7-DMP, and DIP. For L = 5,6-DMP,
an isocratic method of 30:70 MeCN:0.1% TFA (aq) was used. For L = bpy, HDPA, and 4,7-
DMP, the purified product was converted to the chloride salt using Sephadex QAE resin
charged with MgCl
2
. For L = 5,6-DMP and DIP, the purified product was converted to the
chloride salt using Dowex 1×2 500-100 mesh ion exchange resin. Purified yields of 30%
(bpy), 10% (HDPA), 10% (4,7-DMP), 23% (5,6-DMP), 33% (DIP).
Characterization of [Rh(bpy)(chrysi)(PPO)](TFA)
2
LCQ-MS (cation):
m/z
calc. 650.1 (M-1H
+
), 325.6 (M
2+
); obs. 650.0, 325.8. UV-Vis (H
2
O):
259nm (59,800 M
−1
cm
−1
), 287nm (43,100 M
−1
cm
−1
), 298nm (37,100 M
−1
cm
−1
), 312nm
(32,000 M
−1
cm
−1
), 435nm (10,000 M
−1
cm
−1
).
1
H NMR (500 MHz, Acetonitrile-
d
3
)
δ
13.44 (br s, 1.2H), 11.89 (br s, 2H), 9.45 (d,
J
= 5.6 Hz, 1H), 9.36 (d,
J
= 5.7 Hz, 0.6H), 8.80
(d,
J
= 8.0, 1.4 Hz, 1H), 8.71 (d,
J
= 5.3 Hz, 0.6H), 8.62 (d,
J
= 8.2 Hz, 0.6H), 8.60-8.54 (m,
2.6H), 8.43-8.26 (m, 8H), 8.26-8.21 (m, 1H), 8.14 (d,
J
= 8.2, 1.5 Hz, 0.6H), 8.06-7.89 (m,
4.8H), 7.85-7.78 (m, 1,6H), 7.77-7.68 (m, 3.2H), 7.68-7.61 (m, 2.2H), 7.60-7.52 (m, 2.6H),
7.31 (d, 0.6H), 7.29-7.21 (m, 2.6H), 1.91 (s, 3H), 1.87 (s, 1.8H), 1.58 (s, 4.8H), purified as a
1:0.6 mixture of diastereomers.
Characterization of [Rh(HDPA)(chrysi)(PPO)](TFA)
2
LCQ-MS (cation):
m/z
calc. 665.2 (M-1H
+
), 333.1 (M
2+
); obs. 665.3, 333.3. UV-Vis (H
2
O):
259nm (60,400 M
−1
cm
−1
), 283nm (45,900 M
−1
cm
−1
), 326nm (18,600 M
−1
cm
−1
), 440nm
(8,500 M
−1
cm
−1
).
1
H NMR (500 MHz, Acetonitrile-
d
3
)
δ
12.49 (br s, 1H), 12.04 (br s,
1H), 8.72 (dd,
J
= 8.0, 1.3 Hz, 1H), 8.50 (d,
J
= 6.0 Hz, 1H), 8.38-8.31 (m, 3H), 8.31-8.23
(m, 2H), 8.20-8.13 (m, 2H), 8.08-8.00 (m, 2H), 7.98 (td,
J
= 8.6, 1.6 Hz, 1H), 7.94-7.81 (m,
4H), 7.69 (m, 3H), 7.51 (ddd,
J
= 7.6, 6.0, 1.4 Hz, 1H), 7.23 (ddd,
J
= 7.4, 6.1, 1.3 Hz, 1H),
7.17 (ddd,
J
= 7.4, 6.2, 1.4 Hz, 1H), 1.78 (s, 3H), 1.56 (s, 3H), purified as a single
diastereomer.
Characterization of [Rh(4,7-DMP)(chrysi)(PPO)](TFA)
2
LCQ-MS (cation):
m/z
calc. 702.2 (M-1H
+
), 351.6 (M
2+
); obs. 702,3, 351.8. UV-Vis (H
2
O):
269nm (106,800 M
−1
cm
−1
), 440nm (11,400 M
−1
cm
−1
).
1
H NMR (500 MHz, Acetonitrile-
d
3
)
δ
13.31 (br s, 0.8H), 11.75 (br s, 2H), 9.50 (d,
J
= 5.4 Hz, 1H), 9.42 (d,
J
= 5.4 Hz,
0.4H), 8.86 (dd,
J
= 5.5, 0.9 Hz, 1H), 8.83 (dd,
J
= 8.0, 1.3 Hz, 1H), 8.73 (d,
J
= 5.4 Hz,
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0.4H), 8.47 (d,
J
= 2.5 Hz, 0.4H), 8.46-8.35 (m, 4.2H), 8.34 (d,
J
= 8.2 Hz, 0.4H), 8.27 (d,
J
= 8.8 Hz, 1H), 8.21-8.17 (m, 1.4H), 8.16 (d,
J
= 8.1 Hz, 0.4H), 8.08 (dd,
J
= 5.4, 1.0 Hz,
1H), 8.04 (d,
J
= 5.4 Hz, 0.4H), 8.00 (dd,
J
= 7.5, 1.7 Hz, 1H), 7.97-7.92 (m, 2.4H), 7.84 (m,
1.8H), 7.77 (m, 1.4H), 7.61-7.51 (m, 5.2H), 7.19-7.15 (m, 0.4H), 7.10-7.03 (m, 2.8H), 3.05
(s, 3H), 3.04 (s, 1.2H), 3.02 (s, 1.2H), 2.99 (s, 3H), 1.95 (s, 3H), 1.92 (s, 1.2H), 1.62 (s, 3H),
1.61 (s, 1.2H), purified as a 1:0.4 mixture of diastereomers.
Characterization of [Rh(5,6-DMP)(chrysi)(PPO)](TFA)
2
LCQ-MS (cation):
m/z
calc. 702.2 (M-1H
+
), 351.6 (M
2+
); obs. 702.3, 351.8. UV-Vis (H
2
O):
267nm (80,600 M
−1
cm
−1
), 280nm (81,700 M
−1
cm
−1
), 438nm (10,500 M
−1
cm
−1
).
1
H
NMR (500 MHz, Acetonitrile-
d
3
)
δ
13.40 (br s, 0.3H), 11.77 (br s, 1H), 9.68 (d,
J
= 5.2 Hz,
1H), 9.59 (d,
J
= 5.1 Hz, 0.3H), 9.06-8.97 (m, 3.9H), 8.84-8.89 (m, 1.3H), 8.43-8.37 (m,
2.6H), 8.34 (d,
J
= 8.2 Hz, 0.3H), 8.29-8.14 (m, 5.2H), 8.02-7.97 (m, 2.3H), 7.96-7.89 (m,
2.6H), 7.83-7.73 (m, 1.6H), 7.57 (td,
J
= 7.4, 1.4 Hz, 2H), 7.55-7.50 (m, 1H), 7.17 (d,
J
= 5.7
Hz, 0.3H), 7.10-7.02 (m, 2.3H), 2.91 (s, 0.9H), 2.90 (s, 0.9H), 2.89 (s, 3H), 2.87 (s, 3H),
1.93 (s, 3H), 1.90 (s, 0.9H), 1.58 (s, 3.9H), purified as a 1:0.3 mixture of diastereomers.
Characterization of [Rh(DIP)(chrysi)(PPO)]Cl
2
LCQ-MS (cation): m/z calc. 826.2 (M-1H+); obs. 826.3. UV-Vis (H
2
O): 267nm (103,000 M
−1
cm
−1
).
1
H NMR (500 MHz, Methanol-
d
4
)
δ
9.74 (dd, J = 5.5, 0.9 Hz, 1H), 9.70 (dd, J =
5.5, 0.8 Hz, 0.5H), 8.89 (m, 1.5H), 8.76 (m, 1.5H), 8.58-8.46 (m, 4.5H), 8.40-8.28 (m, 6H),
8.14-7.98 (m, 4.5H), 7.81-7.59 (m, 15H), 7.56-7.49 (m, 1.5H), 7.41-7.33 (m, 6H), 7.34-7.23
(m, 3H), 2.07 (s, 3H), 2.02 (s, 1.5H), 1.70 (s, 1.5H), 1.69 (s, 3H), purified as a 1:0.5 mixture
of diastereomers.
Enantiomeric Separation of [Rh(phen)(chrysi)(PPO)]Cl
2
Purified [Rh(phen)(chrysi)(PPO)][TFA]
2
was dissolved in 1:1 ethanol:water and HPLC
purified on an Astec CYCLOBOND chiral column using an isocratic elution method of
40:60 ACN:0.1 M KPF
6
(aq) over 37 min. The column was periodically rinsed with 40:60
MeCN:H
2
O to remove KPF
6
buildup. Separated enantiomers were collected and exchanged
to the chloride salt using Sephadex QAE resin pre-equilibrated with MgCl
2
. The
enantiomeric nature of the collected fractions was verified using circular dichroism (CD) as
follows: 200 μM solutions of Δ- and
Λ
-[Rh(phen)(chrysi)(PPO)]Cl
2
were made in aqueous
solution and their CD spectra recorded in 1 nm increments on an Aviv 62DS
spectropolarimeter under a N
2
atmosphere at ambient temperature. The spectra were
recorded a second time 30 d later to assess decomposition or racemization of the sample,
and none was observed.
Determination of Extinction Coefficients
Aqueous solutions of each [Rh(L)(chrysi)(PPO)]Cl
2
complex were made and a UV-Visible
spectrum was recorded for each. The solutions were diluted 50x, 100x, 500x, and 1000x in
2% HNO
3
. The dilutions were analyzed for Rh content
via
ICP-MS (inductively coupled
plasma mass spectrometry) and the concentration was determined by comparison to a
standard curve. Extinction coefficients were determined from the UV-Visible absorbance
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measurement of the initial solution and the Rh concentration of the dilutions following
Beer’s law (A=
ε
lc). L = DIP was observed to significantly adsorb onto plastics, therefore
PTFE (polytetrafluoroethylene) and PFA (perfluoroalkoxy alkane) coated materials were
used in the workup and analysis of its extinction coefficient.
Partition Coefficient Determination
One-octanol and 10 mM Tris-HCl, pH 7.4 were pre-equilibrated with each other by
vigorously shaking the phases together. A solution of each metalloinsertor was made in
octanol and the UV-Visible spectrum of the solution recorded. Each solution was combined
with an equal volume of aqueous buffer and shaken using a foam insert on a Vortex-Genie 2
running at maximum speed for 16 h. The samples were centrifuged to separate the aqueous
and octanol phases and a UV-Visible spectrum of each octanol fraction was recorded. The
baseline value obtained at 800 nm was used to normalize the spectra to a common zero
point. The absorbance of the ~260 nm peak in the final spectrum was compared to the initial
spectrum to determine the partition coefficient following the literature.
24
The partition
coefficients from three experiments were measured for each [Rh(L)(chrysi)(PPO)]Cl
2
complex and averaged to give the partition coefficient.
pK
a
Determination of Metalloinsertors
A ~25 μM solution of each metalloinsertor was made in 100 mM NaCl. The pH of the
sample was adjusted to 4.5 using HCl (10 mM). NaOH (10 mM) was titrated into the
solution, with stirring. The pH and UV-Visible spectrum were recorded after each base
addition, up to a pH of 10.5. A back titration to pH 6 was performed to check for
decomposition, and none was observed. Spectra were corrected for baseline and volume
changes. The absorbance of the ~430 nm peak was plotted against pH and fit to a sigmoidal
curve in OriginPro v8.5, and the pK
a
was determined as the inflection point of the curve.
Three pK
a
titrations were performed for each [Rh(L)(chrysi)(PPO)]Cl
2
complex and
averaged to give an average pK
a
value.
Binding Constant Determination
A DNA hairpin (5
′
-GGCAGG
X
ATGGCTTTTTGCCAT
Y
CCTGCC-3
′
, where
XY
=CG or
CC for a well-matched or mismatched hairpin, respectively) was radiolabeled with
γ
-
32
P
ATP and prepared following the literature.
10
,
19
,
22
Full details of DNA preparation and
purification can be found in the SI. A 4 μM solution of the photocleaving metalloinsertor
[Rh(bpy)
2
(chrysi)]Cl
3
and solutions containing 0–400 μM of a competing metalloinsertor,
[Rh(L)(chrysi)(PPO)]Cl
2
(which does not photocleave DNA), were made in MilliQ water.
Five μL of the [Rh(bpy)
2
(chrysi)]Cl
3
solution, 5 μL of the competing metalloinsertor, and 10
μL of the hairpin DNA were combined to create a solution containing 1 μM
[Rh(bpy)
2
(chrysi)]Cl
3
, 0–100 μM competing metalloinsertor, and 1 μM DNA. The samples
were irradiated with an Oriel 1000 W Hg/Xe solar simulator (340–440 nm) for 20 min. After
irradiation, solvent was removed from the samples and the samples were counted on a
scintillation counter to determine the necessary exposure time (with 300,000 cpm needing a
1 hour exposure) and they were suspended in a denaturing formamide loading dye. Samples
were electrophoresed on a 20% denaturing polyacrylamide urea gel.
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A phosphor screen was exposed to the polyacrylamide gel and imaged using a Typhoon FLA
9000 biomolecular imager. The ratio of photocleaved to uncleaved DNA was quantified
using ImageQuant TL software. The ratio was plotted against the concentration of [Rh(L)
(chrysi)(PPO)]Cl
2
and fit to a sigmoidal curve in OriginPro v8.5 to determine the inflection
point of the fit. The binding affinity of the competing metalloinsertor was calculated in
Mathematica 9.0 by solving simultaneous equilibria involving DNA, [Rh(bpy)
2
(chrysi)]Cl
3
,
and [Rh(L)(chrysi)(PPO)]Cl
2
. Three photocleavage titrations were performed for each
[Rh(L)(chrysi)(PPO)]Cl
2
complex and averaged to give the binding affinity.
Melting Temperature Analysis
Melting temperature analysis was performed on a Beckman DU 7400 spectrophotometer
equipped with a Tm Analysis Accessory. The short oligomer, 5
′
-CGGA
CTCCG-3
′
(underline denotes mismatch), was purchased from IDT DNA and purified by HPLC.
Samples containing 11 μM ssDNA (ultimately 5.5 μM dsDNA and mismatches) and 6 μM of
[Rh(phen)(chrysi)(PPO)]Cl
2
, [Rh(bpy)
2
(chrysi)]Cl
3
or no metal complex were prepared in
phosphate buffer (5 mM phosphate, 50 mM NaCl, pH 7.0). Samples were heated at a rate of
0.5 °C/min and absorbance was measured at 260 nm every 0.5 °C between 10 °C and 50 °C.
Data from three experiments was combined and fit to a sigmoidal curve in OriginPro v8.5
and the T
m
was taken as the inflection point of the curve.
Cell Culture
HCT116N and HCT116O cells were grown in RPMI (Roswell Park Memorial Institute)
1640 media supplemented with 10% FBS (fetal bovine serum), 2 mM L-glutamine, 0.1 mM
non-essential amino acids, 1 mM sodium pyruvate, 100 units/mL penicillin and
streptomycin, and 100 μg/mL Geneticin (G418). The cells were incubated in tissue culture
flasks or plates at 37 °C in a 5% CO
2
atmosphere. All cell studies were performed with the
chloride salt of each metalloinsertor.
Cell Proliferation ELISA
Cell proliferation ELISA (enzyme-linked immunosorbent assay) was performed following
the manufacturers instructions. Briefly, 2×10
3
HCT116N or HCT116O cells in 100 μL
media were plated into each well of a 96-well plate. The cells were allowed to adhere for 24
h before the addition of 100 μL of media containing various concentrations of rhodium
metalloinsertor. The plates were incubated for an additional 48 h before the rhodium-
containing media was replaced with fresh media, with which the cells were allowed to grow
for the remainder of a 72 h period. Cells were then treated with an excess of the unnatural
nucleic acid, BrdU (bromodeoxyuridine), for 24 h during which time it could be
incorporated into newly synthesized DNA. Cells were then fixed, labeled with a BrdU
antibody, and quantified using a colorimetric substrate solution and stop solution.
Absorbance was measured at 450 nm (background subtracted at 690 nm). Decrease in
cellular proliferation was determined for each metalloinsertor concentration through
comparison to untreated cells. Outliers were removed using a modified Thompson Tau test.
An additional variation of this assay was performed in which the cells were treated with
rhodium metalloinsertor for 24 h, then directly treated with BrdU in fresh media.
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MTT Cytotoxicity Assay
Cell proliferation MTT (MTT = 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltatrazolium
bromide) assays were performed following the manufacturers instructions. Briefly, 5×10
4
HCT116N or HCT116O cells in 100 μL media were plated into each well of a 96-well plate.
Various concentrations of a rhodium metalloinsertor were added to each well. The cells were
allowed to incubate for 72 h before treatment with MTT for 4 h, during which time MTT
could be converted into formazan by metabolically active cells. The formazan crystals were
solubilized and quantified by absorbance at 570 nm (background subtracted at 690 nm).
Viability was determined for each metalloinsertor concentration through comparison to
untreated cells. Outliers were removed using a modified Thompson Tau test. An additional
variation of this assay was performed in which the cells were allowed to adhere to the 96-
well plate overnight before treated with rhodium metalloinsertor for 24 h, followed by MTT
treatment.
Uptake and Localization Experiments
Whole-cell uptake, mitochondrial localization, and nuclear localization of metalloinsertors
were determined following published methods.
25
Prior to whole-cell, mitochondrial, and
nuclear rhodium determination, 24-hour ELISA and MTT assays were performed to
determine a metalloinsertor concentration that would not result in significant cell death by
MTT but showed some anti-proliferative effect by ELISA. The concentrations used in the
uptake and localization studies of the [Rh(L)(chrysi)(PPO)]Cl
2
family were 0.2 μM for
L=DIP, 0.5 μM for L=phen, bpy, HDPA, 4,7-DMP, and 5,6-DMP, and 10 μM for
[Rh(bpy)
2
(chrysi)]Cl
3
, which was included as a control.
Assay for Whole-Cell Rhodium Concentration
Whole-cell uptake experiments were performed following published protocols.
20
Briefly,
1×10
6
HCT116N or HCT116O cells were plated into 6-well tissue culture treated plates and
allowed to adhere for 24 h. Media was aspirated from the cells and fresh media containing a
metalloinsertor was added to each well. Cells were allowed to incubate for an additional
0.5–24 h with the Rh-containing media. After incubation, media was aspirated and the cells
were rinsed with PBS (phosphate buffered saline, pH 7.4) to remove surface rhodium. Cells
were lysed directly in the well using 1 mL of 1% SDS solution. These samples were
transferred to microcentrifuge tubes and sonicated for 10 s at 20% amplitude on a Qsonica
Ultrasonic sonicator. Cell lysate was combined with an equal volume 2% HNO
3
. This
solution was analyzed for Rh content on an Agilent 8800 Triple Quadrupole ICP-MS and the
concentration of Rh in each sample was determined by comparison to a standard curve
(ranging from 1–100 ppb Rh) and normalized using the protein content of each sample. The
protein content of each sample was determined using a Pierce BCA assay, following the
manufacturer’s instructions.
Assay for Mitochondrial Rhodium Concentration
Mitochondrial uptake experiments were performed following published protocols.
20
,
26
Briefly, 1.5×10
7
HCT116N and HCT116O cells were plated in T75 tissue culture treated
flasks. The cells were allowed to adhere for 24 h, after which media was aspirated from each
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flask and restored with 20 mL media containing a rhodium metalloinsertor. The cells were
allowed to grow in the presence of Rh-containing media for 24 h, then harvested using
0.05% trypsin over 5 minutes. Cells were pelleted by centrifugation at 1200 rpm for 5 min.
The pellet was rinsed and suspended in PBS, then pelleted again and the PBS removed. The
cell pellet was suspended in 500 μL mitochondrial extraction buffer (200 mM mannitol, 68
mM sucrose, 50 mM PIPES, 50 mM KCl, 5 mM EGTA, 2 mM MgCl
2
, 1 mM DTT added
just before use, and protease inhibitors added just before use) and incubated on ice for 20
min. Each sample was homogenized by 35 passes thorough a 21-gauge needle and syringe.
The resultant solution was centrifuged for 5 min at 750 rpm. The supernatant of each sample
was transferred to a 1.5 mL microcentrifuge tube and centrifuged for 10 min at 14,000 g.
The supernatant was decanted and the resulting pellet was the mitochondrial fraction. SDS
(800 μL of a 1% solution) was added to the pellet and sonicated for 10 s at 40% amplitude
on a Qsonica Ultrasonic sonicator. Mitochondrial lysate was combined with an equal volume
of 2% nitric acid. This solution was analyzed for Rh content on an Agilent 8800 Triple
Quadrupole ICP-MS and the concentration of Rh in each sample was determined by
comparison to a standard curve (ranging from 1–100 ppb Rh) and normalized using the
protein content of each sample. The protein content of each sample was determined using a
Pierce BCA assay, following the manufacturer’s instructions.
Assay for Nuclear Rhodium Concentration
Nuclear uptake experiments were performed following published protocols.
20
Briefly, 1×10
7
HCT116N and HCT116O cells were plated in T75 tissue culture treated flasks. The cells
were allowed to adhere for 24 h before the media was aspirated and restored with 20 mL
media containing a rhodium metalloinsertor. The cells were allowed to grow in the presence
of Rh-containing media for 24 h, then harvested using 0.05% trypsin over 5 minutes. Cells
were pelleted by centrifugation at 1200 rpm for 5 min. The pellet was rinsed and suspended
in PBS, then pelleted and the PBS removed. Each cell pellet was suspended in 1 mL
hypotonic buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl
2
), transferred to a
microcentrifuge tube, and incubated on ice for 15 min. NP-40 (50 μL of a 10% solution) was
added to each sample, vortexed for 10 s at the highest setting, and centrifuged at 3000 g for
10 min. The supernatant was decanted and the resulting pellet was the nuclear fraction. SDS
(800 μL of a 1% solution) was added to the pellet and then sonicated for 10 s at 40%
amplitude on a Qsonica Ultrasonic sonicator. Nuclear lysate was combined with an equal
volume of 2% HNO
3
. This solution was analyzed for Rh content on an Agilent 8800 Triple
Quadrupole ICP-MS and the concentration of Rh in each sample was determined by
comparison to a standard curve (ranging from 1–100 ppb Rh) and normalized using the
protein content of each sample. The protein content of each sample was determined using a
Pierce BCA assay, following the manufacturer’s instructions.
Assay for Uptake of Metalloinsertors
Mechanism of uptake experiments were adapted from published protocols.
27
RbCl and
[Ru(DIP)(dppz)]Cl
2
were used as positive and negative controls, respectively. Briefly, 1×10
6
HCT116N or HCT116O cells were plated into 6-well tissue culture treated plates and
allowed to adhere for 24 h. Metabolic inhibitors (5 μM oligomycin in ethanol and 50 mM 2-
deoxy-D-glucose) or control solutions (5 mM glucose and ethanol) were added to the cell
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culture media and samples were incubated for 1 h. Media was removed by aspiration and
each well was washed with PBS. Media (3 mL) containing the
Rh–O
metalloinsertor
[Rh(phen)(chrysi)(PPO)]Cl
2
(0.5 μM), the parent metalloinsertor [Rh(bpy)
2
(chrysi)]Cl
3
(10
μM), [Ru(DIP)(dppz)]Cl
2
(2 μM), or RbCl (25 μM) was then added to each well and
incubated for 1 h. Media was aspirated and cells were rinsed with PBS to remove surface
rhodium, ruthenium, or rubidium. Cells were lysed directly in the well using 1 mL of 1%
SDS solution. Samples were transferred to microcentrifuge tubes and sonicated for 10 s at
20% amplitude on a Qsonica Ultrasonic sonicator. Cell lysate was combined with an equal
volume of 2% HNO
3
and analyzed for Rh, Ru, and Rb content on an Agilent 8800 Triple
Quadrupole ICP-MS, and the concentration of Rh, Ru, or Rb in each sample was determined
by comparison to a standard curve (ranging from 1–100 ppb) and normalized using the
protein content of each sample. The protein content of each sample was determined using a
Pierce BCA assay, following the manufacturer’s instructions.
RESULTS
Establishing the Enantiomeric Activity of [Rh(phen)(chrysi)(PPO)]
2+
Enantiomeric separation was performed for the complex [Rh(phen)(chrysi)(PPO)]
2+
to
establish the interaction of its Δ- and
Λ
-enantiomers with DNA
in vitro
and in MMR-
deficient or -proficient cells in culture. The Δ- and
Λ
- enantiomers of [Rh(phen)(chrysi)
(PPO)]
2+
were isolated with >90% and >95% enantiomeric excess, respectively
(Supplementary Figure S1). Circular dichroism experiments confirmed the enantiomeric
nature of the isolated complexes, and no racemization was observed at ambient temperature
over 1 month (Supplementary Figure S1). Competition titrations between [Rh(phen)(chrysi)
(PPO)]
2+
and the photocleaving metalloinsertor [Rh(bpy)
2
(chrysi)]
3+
in the presence of
32
P-
radiolabeled DNA containing a CC mismatch revealed both enantiomers are capable of
binding mismatched DNA base pairs with similar affinity (10
6
M
−1
, Table 1).
10
Furthermore, both enantiomers were found to have selective cytotoxic effects towards
MMR-deficient cells over MMR-proficient cells in MTT experiments (Supplementary
Figure S2). These studies confirm that both enantiomers of the PPO-containing
metalloinsertor, [Rh(phen)(chrysi)(PPO)]
2+
, exhibit binding properties towards mismatched
DNA that are consistent with a previous generation of
Rh–O
metalloinsertors. These
Rh–O
complexes show no enantiomeric preference in binding DNA, unlike parent metalloinsertors,
which show a high enantiomeric preference for the Δ-isomer in binding DNA.
15
,
19
Binding of Metalloinsertors to a Single Base Pair Mismatch
The binding affinities of [Rh(L)(chrysi)(PPO)]
2+
metalloinsertors to DNA containing a
single CC mismatch were determined. The [Rh(L)(chrysi)(PPO)]
2+
complexes do not
photocleave DNA upon irradiation, so their binding affinities were assayed
via
a competition
titration with [Rh(bpy)
2
(chrysi)]
3+
, a complex known to photocleave DNA selectively upon
mismatch binding and irradiation.
22
A CC mismatch was used as it is highly destabilized
relative to other mismatches and therefore undergoes significant photocleavage in the
presence of [Rh(bpy)
2
(chrysi)]
3+
. A constant concentration of [Rh(bpy)
2
(chrysi)]
3+
and
varying concentrations of the competing [Rh(L)(chrysi)(PPO)]
2+
metalloinsertor were
incubated with a DNA hairpin containing a single CC mismatch, irradiated, and the DNA
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photocleavage products were separated on a denaturing gel. The ratio of photocleaved DNA
to intact DNA was plotted against the log of the rhodium concentration and fit to a sigmoidal
curve (Figure S3). The inflection point of the sigmoidal fit was used to determine the
binding affinity of the competing [Rh(L)(chrysi)(PPO)]
2+
metalloinsertor by solving
simultaneous equilibria equations using the known binding affinity of [Rh(bpy)
2
(chrysi)]
3+
.
The binding affinities of these complexes are shown in Table 1. All complexes were tested as
racemic mixtures and exhibit binding affinities in the range of 2.4 to 7.2 × 10
6
M
−1
(Table
1). Despite differences in ligand steric bulk, all
Rh–O
metalloinsertors tested have binding
affinities within one order of magnitude of each other, and thus bind DNA with comparable
affinity.
Binding was assessed further via melting temperature analysis. A short, palindromic DNA
sequence containing a central CC mismatch was incubated in the presence of the parent
metalloinsertor, [Rh(bpy)
2
(chrysi)]Cl
3
, or the
Rh–O
metalloinsertor, [Rh(phen)(chrysi)
(PPO)]Cl
2
. The chosen DNA sequence has a low T
m
and therefore exists as ssDNA at room
temperature.
13
In the presence of metalloinsertor, however, the DNA anneals and the melting
temperature increases dramatically to 44.9 ± 0.6 and 41.3 ± 0.5 °C for [Rh(bpy)
2
(chrysi)]Cl
3
and [Rh(phen)(chrysi)(PPO)]Cl
2
, respectively (Supplementary Figure S3B). These results
are in good agreement with the results of the DNA binding assay describe above and
corroborate the result that parent and
Rh–O
metalloinsertors have comparable binding
affinities to mismatches in DNA, with [Rh(phen)(chrysi)(PPO)]Cl
2
stabilizing DNA to a
slightly lesser extent than [Rh(bpy)
2
(chrysi)]Cl
3.
pK
a
Determination of Metalloinsertors
The pK
a
values of [Rh(L)(chrysi)(PPO)]
2+
metalloinsertors were assessed
via
spectroscopic
pH titrations (Table 1, Supplementary Figures S4–S8). The absorbance of a 435–440 nm
peak, which corresponds to a charge transfer located on the chrysi ligand, was plotted
against the pH of the solution for each complex.
28
Data were fit to a sigmoidal curve and the
inflection point was taken as the pK
a
of the complex, specifically of the imine proton on the
chrysi ligand. All
Rh–O
metalloinsertors exhibited pK
a
values in the range of 8.1 to 9.1,
which are above physiological pH (Table 1), indicating that the chrysi ligands of these
complexes remain protonated in cell culture media or within cells. It has been shown
previously that fully protonated chrysi ligands, which are seen with
Rh–O
metalloinsertors,
buckle in contrast to the deprotonated chrysi ligands of the parent metalloinsertors, which
are completely flat and thus easy to stack with the DNA base pairs once inserted.
19
Partition Coefficient and Lipophilicity of Metalloinsertors
The [Rh(L)(chrysi)(PPO)]Cl
2
family of metalloinsertors was designed to vary in
lipophilicity, and the partition coefficients of each [Rh(L)(chrysi)(PPO)]
2+
metalloinsertor
were determined between aqueous buffer (10 mM Tris-HCl, pH 7.4) and 1-octanol
according to literature methods.
24
Absorbance measurements at the ~260 nm peak were
made in the 1-octanol phase before and after equilibration with the aqueous phase. These
absorbance values were compared to determine the partition coefficient, log P (Table 1,
Supplementary Figures S9–S12). The log P values followed the expected trend with the least
bulky complexes ([Rh(bpy)(chrysi)(PPO)]
2+
and [Rh(HDPA)(chrysi)(PPO)]
2+
) having the
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lowest log P values and the bulkiest complex ([Rh(DIP)(chrysi)(PPO)]
2+
) having the
greatest log P value. Surprisingly, despite their cationic nature, under these conditions the
[Rh(L)(chrysi)(PPO)]
2+
metalloinsertors are all lipophilic and have partition coefficients
favoring octanol over water, ranging from 0.68 to >2.0.
Cytotoxic and Anti-Proliferative Effects in MMR-Deficient and -Proficient Cells
The ability of metalloinsertors to selectively kill or impair growth of MMR-deficient cells is
a critical factor in their potential value as chemotherapeutic agents.
19
,
29
In this structure-
activity relationship study, we used ELISA and MTT assays to determine the effect of ligand
substitution on biological activity in MMR-deficient and -proficient cells. The ELISA was
used to determine the inhibitory effects on DNA synthesis and the MTT assay was
performed to establish levels of cytotoxicity. For the ELISA, each metalloinsertor was
incubated with HCT116N (MMR-proficient) or HCT116O (MMR-deficient) cells at various
concentrations before treatment with the unnatural nucleic acid BrdU. Colorimetric antibody
treatment allowed the relative BrdU incorporation into DNA to be quantified, and cellular
proliferation was then determined as the ratio of BrdU incorporation between
metalloinsertor-treated cells and untreated control cells. The results of the 48-hour
metalloinsertor treatment are shown in Figure 3, and the results of a 24-hour treatment are
shown in Supplemental Figure S15. All [Rh(L)(chrysi)(PPO)]
2+
metalloinsertors exhibit
anti-proliferative activity with selectivity towards the MMR-deficient cell line. The
maximum proliferation difference (referred to as selectivity) between the cell lines and the
concentration at which this selectivity occurs (referred to as potency) are as follows: 77
± 10% at 400 nM for [Rh(phen)(chrysi)(PPO)]
2+
, 78 ± 18% at 2 μM for [Rh(bpy)(chrysi)
(PPO)]
2+
, 47 ± 10% at 25 μM for [Rh(HDPA)(chrysi)(PPO)]
2+
, 66 ± 6% at 400 nM for
[Rh(4,7-DMP)(chrysi)(PPO)]
2+
, 67 ± 5% at 400 nM for [Rh(5,6-DMP)(chrysi)(PPO)]
2+
,
and 70 ± 23% at 160 nM for [Rh(DIP)(chrysi)(PPO)]
2+
.
For the MTT assay, each metalloinsertor was incubated with HCT116N (MMR-proficient)
or HCT116O (MMR-deficient) cells at various concentrations before the addition of MTT,
which can be converted into formazan by mitochondrial reductase activity in a functioning
cell. Colorimetric measurements of formazan allow the relative viability to be quantified,
and cellular viability is then determined as the ratio of formazan produced between
metalloinsertor-treated cells and untreated control cells. The results of the 72-hour treatment
are shown in Figure 4 and the results of the 24-hour treatment are shown in Supplemental
Figure S16. All [Rh(L)(chrysi)(PPO)]
2+
metalloinsertors exhibit cytotoxic activity with
selectivity towards the MMR-deficient cell line. The maximum proliferation difference
between the cell lines and the concentration at which this difference occurs are as follows:
52 ± 5% at 300 nM for [Rh(phen)(chrysi)(PPO)]
2+
, 30 ± 7% at 2 μM for [Rh(bpy)(chrysi)
(PPO)]
2+
, 13 ± 11% at 32 μM for [Rh(HDPA)(chrysi)(PPO)]
2+
, 46 ± 8% at 600 nM for
[Rh(4,7-DMP)(chrysi)(PPO)]
2+
, 49 ± 3% at 600 nM for [Rh(5,6-DMP)(chrysi)(PPO)]
2+
,
and 39 ± 6% at 640 nM for [Rh(DIP)(chrysi)(PPO)]
2+
.
Whole-Cell Uptake, Mechanism of Uptake, and Organelle Localization
To better understand the range of biological activities of these complexes, cellular uptake
and mechanism of uptake were examined
via
ICP-MS based assays. 24-hour ELISA and
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MTT assays were performed to determine a suitable concentration for uptake and
localization studies (which were performed over a 24-hour timescale). To minimize cell
death in this assay, a factor which can complicate data interpretation, suitable dosing was
determined to be at a concentration at which there was noticeable anti-proliferative effects in
the HCT116O cells
via
ELISA but no significant cytotoxicity
via
MTT assay. Whole cell
uptake studies were performed with each [Rh(L)(chrysi)(PPO)]
2+
complex at 0.5 μM with
the exception of [Rh(DIP)(chrysi)(PPO)]
2+
, which was performed at 0.2 μM due to its high
cytotoxicity at 0.5 μM. For whole cell uptake studies, cells were incubated with
metalloinsertors for 24 h before they were lysed and analyzed for rhodium content
via
ICP-
MS, with rhodium concentrations normalized to the protein content of each sample. The
whole cell uptakes of each metalloinsertor in HCT116O cells are shown in Figure 5 (results
in HCT116N cells are similar and shown in Supplementary Figure S17). Overall, all [Rh(L)
(chrysi)(PPO)]
2+
complexes exhibit uptake into cells at concentrations within one order of
magnitude of each other. The uptake of these complexes correlates generally with their
lipophilicity values, with the least lipophilic complexes ([Rh(HDPA)(chrysi)(PPO)]
2+
and
[Rh(bpy)(chrysi)(PPO)]
2+
) having the poorest uptake and the most lipophilic complex
([Rh(DIP)(chrysi)(PPO)]
2+
) having the highest uptake. Lipophilicity has long been
correlated with an increase in cellular uptake and a resultant increase in drug potency.
30
,
31
In addition to examining whole cell uptake of the [Rh(L)(chrysi)(PPO)]
2+
metalloinsertors,
the uptake over time and the mechanism of uptake were also examined. In the former
experiment, cells were incubated with a metalloinsertor for 0.5, 1, 3, 6, 9, or 24 h before
being lysed and analyzed for rhodium content by ICP-MS. The whole-cell uptake over time
of these metalloinsertors in HCT116O cells is shown in Figure 5 (results in HCT116N cells
are similar and shown in Supplementary Figure S17). The complexes appear to show
significant increases in uptake over the first 3–6 h of incubation with cells, followed by
plateau with no evidence of significant efflux during a 24-hour period. These results are
consistent with previous studies on metalloinsertors.
20
A metabolic inhibition assay was performed to better understand the mechanism of cellular
uptake of [Rh(L)(chrysi)(PPO)]
2+
metalloinsertors. HCT116N and HCT116O cells were
pre-treated with the metabolic inhibitors oligomycin A, an inhibitor of oxidative
phosphorylation, and 2-deoxy-D-glucose, an inhibitor of glycolysis.
27
Metabolic inhibition
depletes cellular ATP (adenosine triphosphate), so any compound that is taken into the cell
via
an active, ATP-dependent mechanism should have reduced uptake in metabolically
depleted cells. Conversely, complexes taken into the cell
via
a passive mechanism, such as
passive diffusion, are not affected by metabolic inhibition and therefore the drug should
accumulate in inhibited and uninhibited cells at similar concentrations. [Rh(phen)(chrysi)
(PPO)]
2+
and the parent metalloinsertor, [Rh(bpy)
2
(chrysi)]
3+
, were studied to determine if
the mechanism of metalloinsertor uptake was ATP-dependent. The compounds RbCl and
[Ru(dppz)(DIP)
2
]
2+
were included as positive and negative controls, respectively. The
rubidium ion of RbCl is transported into the cell by Na,K-ATPase, an ATP-dependent ion
pump, while [Ru(dppz)(DIP)
2
]
2+
has previously been shown to enter the cell
via
passive
diffusion.
27
,
32
Cells were treated with each compound for 1 h before they were lysed and
analyzed by ICP-MS for metal content. As rubidium, ruthenium, and rhodium are not
naturally present in cells or cell culture reagents, all three elements can be analyzed as low-
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background analytes by ICP-MS. The results of each compound in HCT116O cells are
shown in Figure 6 (results in HCT116N cells are similar and shown in Supplementary
Figure S18). As expected, RbCl showed a significant decrease in uptake when pre-treated
with metabolic inhibitors and [Ru(dppz)(DIP)
2
]
2+
was unaffected by inhibitor pre-treatment.
Similar to [Ru(dppz)(DIP)
2
]
2+
, [Rh(phen)(chrysi)(PPO)]
2+
and [Rh(bpy)
2
(chrysi)]
2+
were
also unaffected by inhibitor pre-treatment, suggesting these complexes are also taken into
the cell via an ATP-independent mechanism, such as passive diffusion. Since these
complexes are all lipophilic and cationic, passive diffusion is a reasonable uptake
mechanism, with the negative membrane potential driving diffusion and relatively high
lipophilicity facilitating the process as the molecules can more readily partition into the
cellular membranes.
33
Subcellular localization into the nucleus (the on-target organelle) and mitochondria (a major
off-target organelle) were also examined by an ICP-MS assay. Localization studies were
performed with each [Rh(L)(chrysi)(PPO)]
2+
metalloinsertor at 0.5 μM with the exception
of [Rh(DIP)(chrysi)(PPO)]
2+
, which was performed at 0.2 μM. For localization studies, cells
were incubated with metalloinsertors for 24 h before they were lysed and analyzed for
rhodium content via ICP-MS, with rhodium concentrations normalized to the protein content
of each sample. The whole cell uptakes of each metalloinsertor in HCT116O cells are shown
in Figure 7 (results in HCT116N cells are similar and shown in Supplementary Figure S19).
Overall, all [Rh(L)(chrysi)(PPO)]
2+
complexes have comparable nuclear uptakes and
mitochondrial uptakes to one another with the exception of [Rh(DIP)(chrysi)(PPO)]
2+
,
which has nuclear and mitochondrial uptakes that are 2–3 times higher than other complexes
despite being dosed at a lower concentration. All complexes appear to enter the nucleus at
high enough concentrations to bind DNA mismatches, with a significant enrichment in
nuclear concentration over the extracellular concentration of rhodium (Supplementary Table
S1).
DISCUSSION
Early generations of rhodium metalloinsertors, which exclusively contain Rh–N ligand
coordination, are a richly studied family of metal complexes that can selectively bind to
DNA base pair mismatches and lead to selective cell death in MMR-deficient cells. Across
multiple studies, these metalloinsertors were determined to have several characteristic and
consistent behaviors. Through
in vitro
experiments, we have observed that only the Δ-
enantiomer of these Rh–N coordinated complexes is capable of binding mismatches in B-
form DNA.
34
In cellular studies, these metalloinsertors have been observed to selectively kill
cells in concentration ranges of 5–40 μM.
20
,
21
In one structure-activity relationship study,
the steric bulk of the ancillary ligands on a metalloinsertor was seen to influence DNA
binding properties and, ultimately, alter cellular selectivity.
21
In another structure-activity
relationship study, the lipophilicity of the ancillary ligands on a metalloinsertor was seen to
dramatically influence its subcellular localization within a cell and, again, alter cellular
selectivity.
20
While the above trends seem to ring true across parent metalloinsertors containing
exclusively Rh–N ligand coordination, the recent emergence of a new family of
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metalloinsertors that contain Rh–O ligand coordination has challenged many of these
characteristics and behaviors.
19
For instance, both enantiomers of
Rh–O
metalloinsertors
are capable of binding DNA mismatches
in vitro
, and are furthermore capable of inducing
selective cellular toxicity at
nanomolar
concentrations. Additionally, changes in lipophilicity
and steric bulk of the O-containing ligand seemed to have little, if any, effect on DNA
binding affinity and cellular selectivity. This remarkable shift in metalloinsertor activity
revealed that these
Rh–O
complexes have distinct
in vitro
characteristics and biological
properties from their parent metalloinsertor complexes. As such, a new family of
Rh–O
metalloinsertors has been synthesized, characterized, and investigated for biological activity.
In contrast to the first generation of
Rh–O
metalloinsertors in which the O-containing ligand
was varied, in this new family an ancillary ligand was varied and the O-containing ligand
was kept constant. This family is of the form [Rh(L)(chrysi)(PPO)]
2+
, where L = bpy, phen,
HDPA, 4,7-DMP, 5,6-DMP, and DIP. This ligand variation influences many features of the
metalloinsertor, including steric bulk and lipophilicity, both of which have previously been
seen to affect DNA binding and cellular activity of the parent metalloinsertors.
20
,
21
In
studying this family of complexes, we aimed to test the unique biological activity of
metalloinsertors containing the
Rh–O
ligand framework and begin to understand the high
potency and improved selectivity exhibited by these metalloinsertors over parent
metalloinsertors and other DNA-binding complexes.
Robustness of Biological Activity of the Rh–O Ligand Framework
A primary aim of this structure-activity relationship study was to determine if altering the
ancillary ligand of
Rh–O
metalloinsertors would significantly affect the biological activity
of these complexes. Biological activity was assessed through both ELISA and MTT assays
in two cell lines, HCT116N and HCT116O. These cells are derived from the same colorectal
carcinoma cell line but differ primarily in that HCT116N cells are MMR-proficient whereas
HCT116O cells are MMR-deficient.
35
For this reason, HCT116O cells have a higher relative
abundance of DNA mismatches over HCT116N cells and therefore should be more sensitive
to mismatch-targeting metalloinsertors.
36
Indeed, all complexes prepared showed highly selective anti-proliferative or cytotoxic effects
toward the MMR-deficient cells over the MMR-proficient cells in both ELISA (Figure 3)
and MTT assays (Figure 4), with the exception of [Rh(HDPA)(chrysi)(PPO)]
2+
, which only
shows activity in the ELISA. While selectivity was seen for all complexes, the effective
concentrations varied by two orders of magnitude across the family. For instance,
[Rh(HDPA)(chrysi)(PPO)]
2+
has very low potency and little selectivity compared to other
Rh–O
metalloinsertors. Although it does appear to interfere selectively with DNA synthesis
via
ELISA, this biological interaction does not appear significant enough to produce
cytotoxic effects in the MTT assay, even at high drug concentrations (Figure 4). HDPA is the
only ligand containing a labile proton and the only ligand that forms a 6-ring chelate with
the metal, and it seems possible that these structural features ultimately influence the
biological activity of the [Rh(HDPA)(chrysi)(PPO)]
2+
. It is possible that the 6-member
chelate could cause structural aberrations and the proton on HDPA could cause hydrogen-
bonding interactions that ultimately alter DNA-binding or DNA-processing by proteins,
which could cause a decrease in toxicity. [Rh(bpy)(chrysi)(PPO)]
2+
has the second lowest
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potency of this new family, though remarkably this complex still shows higher potency than
the parent metalloinsertors containing only Rh–N coordination.
20
The phenanthroline-
derived metalloinsertors, [Rh(phen)(chrysi)(PPO)]
2+
, [Rh(4,7-DMP)(chrysi)(PPO)]
2+
, and
[Rh(5,6-DMP)(chrysi)(PPO)]
2+
, all show comparable nanomolar potencies and selectivities
in the ELISA and MTT assays.
Perhaps the most surprising biological activity is seen with [Rh(DIP)(chrysi)(PPO)]
2+
.
Historically, metalloinsertors containing the bulky DIP ligand have shown no selectivity for
the MMR-deficient cell line.
21
This lack of selectivity was attributed to substantially lower
mismatch binding affinities (10
4
M
−1
for [Rh(DIP)
2
(chrysi)]
3+
) owing to ancillary bulk, as
well as off-target localization into the mitochondria, a property that is common with
lipophilic cations.
20
,
37
[Rh(DIP)(chrysi)(PPO)]
2+
, however,
does
exhibit selective
cytotoxicity towards MMR-deficient cells over proficient cells in both the ELISA and MTT
assays. In fact, [Rh(DIP)(chrysi)(PPO)]
2+
displays a similar selectivity and ~2-fold higher
potency than [Rh(phen)(chrysi)(PPO)]
2+
when measured by ELISA (Figure 3).
Overall, these results confirm that
Rh–O
metalloinsertor biological selectivity is minimally
influenced by substitution at the ancillary ligand.
19
Thus far, all of the
Rh–O
metalloinsertors, derivatized at the O-containing ligand or ancillary ligand, have exhibited
selectivity in ELISA and/or MTT assays, regardless of steric bulk or lipophilicity, factors
that had heavily influenced (and sometimes abolished) the selectivity of parent
metalloinsertors. It is noteworthy that this selectivity profile, wherein the
Rh–O
metalloinsertors selectively kill MMR-deficient cells, is shared with the parent complexes
and is in stark contrast to what is seen with all other DNA-targeting therapeutics, which
preferentially kill MMR-proficient cells.
17
,
18
Although parent and
Rh–O
metalloinsertors
share this unique selectivity profile and have similar
in vitro
binding properties, suggesting
they should interact with DNA in a similar way, the
Rh–O
metalloinsertors are dramatically
more potent than the parent metalloinsertors, with nearly all
Rh–O
complexes (with the sole
exception being [Rh(HDPA)(chrysi)(PPO)]
2+
) having greater cytotoxicity in MMR-deficient
cells than
any
of the parent metalloinsertors. It stands to reason, then, that the high potency
and selectivity of these
Rh–O
complexes does not reflect a difference in DNA binding
affinity from the parent complexes, but rather it must instead reflect a difference in structure
associated with the DNA-metalloinsertor lesion. That is, if the frequency of DNA binding is
comparable between the
Rh–O
and parent metalloinsertors, the lesion formed by
Rh–O
metalloinsertors must activate a cellular response at lower concentrations.
Uptake Characteristics
Although the [Rh(L)(chrysi)(PPO)]
2+
family shows consistent activity towards MMR-
deficient cells, the selectivities and potencies of these complexes vary significantly across
the family from 160 nM to 25 μM. It was initially hypothesized that these differences in
biological activity could be due to differences in cellular uptake. In particular, it seemed
possible that the least potent complexes, [Rh(HDPA)(chrysi)(PPO)]
2+
(which has almost no
cytotoxic properties at 40 μM) and [Rh(bpy)(chrysi)(PPO)]
2+
(which has nearly 10-fold
lower potency than [Rh(phen)(chrysi)(PPO)]
2+
), could be less effective due to low uptake.
Similarly, it was proposed that increased uptake could be responsible for the high potency of
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[Rh(DIP)(chrysi)(PPO)]
2+
. Indeed, it does seem possible that uptake may explain some of
the observed potency trends: despite being dosed at 0.2 μM, [Rh(DIP)(chrysi)(PPO)]
2+
exhibits similar uptake to [Rh(phen)(chrysi)(PPO)]
2+
, which was dosed at 0.5 μM. The
finding suggests that [Rh(DIP)(chrysi)(PPO)]
2+
may induce biological effects at half the
concentration of [Rh(phen)(chrysi)(PPO)]
2+
as a result of complexes exhibiting similar
uptakes at these concentrations. However, uptake alone appears insufficient to explain the
potencies of other complexes. For instance, [Rh(HDPA)(chrysi)(PPO)]
2+
and [Rh(bpy)
(chrysi)(PPO)]
2+
have comparably low uptake into the cell despite a >10-fold difference in
activity.
Organelle-specific uptake is also worthy of consideration when examining the activity of
these complexes. Studies on previous generations of parent metalloinsertors bearing solely
Rh–N ligand coordination showed that off-target mitochondrial uptake is strongly influenced
by ligand lipophilicity, with the most lipophilic parent metalloinsertors having high
mitochondrial uptake and low selectivity for MMR-deficient cells.
20
,
38
Surprisingly,
all
Rh–
O
metalloinsertors studied here are more lipophilic than
any
of the parent metalloinsertors
described above, yet all
Rh–O
complexes exhibit selective cytotoxicity towards MMR-
deficient cells, making their selectivity patterns distinct from trends followed by the parent
metalloinsertors. To better understand this marked change in trends, on-target nuclear
localization and off-target mitochondrial localization experiments were performed to assess
the biological activity of [Rh(L)(chrysi)(PPO)]
+2
complexes, particularly DIP, which shows
selectivity despite its very high lipophilicity.
As indicated, all [Rh(L)(chrysi)(PPO)]
2+
metalloinsertors enter the nuclei to a similar extent
and at high enough concentrations to bind DNA mismatches (Figure 7 and Supplementary
Table S1). Similarly, all [Rh(L)(chrysi)(PPO)]
2+
metalloinsertors enter the mitochondria to a
comparable extent. Although nuclear and mitochondrial uptake cannot be compared directly
(since each is normalized to the total protein in the organelle), the localization patterns of
Rh–O
versus parent metalloinsertors can be compared (See Supplementary Figure S20).
This comparison shows that, unlike their Rh–N coordinated predecessors,
Rh–O
metalloinsertor localization into the mitochondria is not significantly influenced by
lipophilicity. In fact, despite being lipophilic,
Rh–O
complexes exhibit uptake profiles that
are comparable to
hydrophilic
parent metalloinsertors (which have low mitochondrial
uptake) and are distinct from
lipophilic
parent metalloinsertors (which have high
mitochondrial uptake). This trend in localization is consistent with the biological activity we
observed; similar to the hydrophilic parent metalloinsertors,
Rh–O
complexes are highly
selective and show little off-target cytotoxicity. Overall, these data indicate that
Rh–O
metalloinsertors are able to maintain their high selectivity and potency because the ligand
substitutions do not strongly influence their subcellular localization. Since these complexes
exhibit low mitochondrial uptake, off-target mitochondria-induced toxicity does not
overwhelm the biological response, and the selective nuclear- and mismatch-mediated
response can prevail.
It is also interesting to note that both MMR-proficient HCT116N cells and MMR-deficient
HCT116O cells had comparable levels of uptake and similar localization profiles, showing
that metalloinsertors enter HCT116N and HCT116O cells at the same rate, through the same
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passive mechanism, and to the same extent (Figure 5 and 6 and Supplementary Figures S17
and S18). These details support the idea that the biological selectivity seen in these cells is
not a feature of different cellular uptake or elimination properties. Furthermore, the nuclear
uptake into the MMR-deficient and proficient cells are comparable (Figure 7 and
Supplementary Figure S19). Therefore, with similar concentrations of metalloinsertors
entering the nuclei and similar mismatch binding affinities, any DNA-mediated cytotoxicity
must result from a difference in how the drugs interact with the DNA. Rationally, this
difference must depend upon an increased mismatch targeting in MMR-deficient cells,
where DNA base pair mismatches are more abundant.
36
Source of Potency for the Rh–O Metalloinsertors
Although MMR-deficient cells have a relative abundance of mismatches compared to MMR-
proficient cells, the total number of mismatches formed during each cellular replication is
ultimately small due to the high fidelity and proofreading abilities of polymerases. It is clear,
therefore, that the lesion formed by parent metalloinsertors must be significantly potent such
that even a small number of metalloinsertor-DNA lesions can result in selective cell death.
Moreover, despite their similar mismatch binding affinities, the
Rh–O
metalloinsertors are
even more potent than parent metalloinsertors, and therefore these
Rh–O
metalloinsertors
must produce a unique lesion structure at the mismatched site that can activate a response at
even lower concentrations (and therefore fewer metalloinsertor-DNA lesions) than parent
metalloinsertors.
Does the increase in potency depend upon a difference in how these
Rh–O
metalloinsertors
bind to DNA within the cell?
19
As discussed above, both the Δ- and
Λ
-enantiomers of
[Rh(phen)(chrysi)(PPO)]
2+
can bind to DNA mismatches
in vitro
and selectively kill MMR-
deficient cells in culture. This behavior is distinct from parent metalloinsertors, for which
only the Δ-enantiomer can bind mismatches and produce biological effects.
15
The ability of
both enantiomers of
Rh–O
metalloinsertors to bind mismatched DNA suggests the binding
interaction must be fundamentally distinct from that of the parent metalloinsertors; these
new
Rh–O
metalloinsertors must bind DNA in a way that can accommodate the
Λ
-
enantiomer.
Furthermore, some evidence suggests that even the DNA-binding ability of the Δ-enantiomer
may be altered in these
Rh–O
metalloinsertors. Previously, it was observed that bulky parent
metalloinsertors, such as [Rh(DIP)
2
(chrysi)]
3+
, exhibited poor binding affinities (10
4
M
−1
)
and could not easily be modeled to fit into a mismatched DNA lesion due to significant
steric clashing between the DIP ligands and the DNA backbone.
21
In contrast, significant
differences in ancillary ligand steric bulk have minimal effect on the binding affinities of
Rh–O
metalloinsertors, which all bind to DNA with micromolar affinity. Even the most
sterically bulky complex, [Rh(DIP)(chrysi)(PPO)]
2+
, has a relatively high affinity for
mismatched DNA (10
6
M
−1
) despite containing the bulky DIP ligand. It therefore seems that
the inclusion of the DIP ligand is not sufficient to preclude DNA binding, and perhaps this
dramatic increase in binding affinity of a DIP-containing metalloinsertor may indicate that a
new binding interaction exists that can accommodate the steric bulk of these
Rh–O
metalloinsertors.
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