of etoposide quinone with topoisomerase ii# a two ...€¦ · 1 a two-mechanism model for the...
TRANSCRIPT
Subscriber access provided by Northern Illinois University
Chemical Research in Toxicology is published by the American Chemical Society.1155 Sixteenth Street N.W., Washington, DC 20036Published by American Chemical Society. Copyright © American Chemical Society.However, no copyright claim is made to original U.S. Government works, or worksproduced by employees of any Commonwealth realm Crown government in the courseof their duties.
Article
A Two-Mechanism Model for the Interactionof Etoposide Quinone with Topoisomerase II#
Elizabeth G. Gibson, McKenzie M. King, Susan L Mercer, and Joseph E. DeweeseChem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00209 • Publication Date (Web): 17 Aug 2016
Downloaded from http://pubs.acs.org on August 23, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are postedonline prior to technical editing, formatting for publication and author proofing. The American ChemicalSociety provides “Just Accepted” as a free service to the research community to expedite thedissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscriptsappear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have beenfully peer reviewed, but should not be considered the official version of record. They are accessible to allreaders and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offeredto authors. Therefore, the “Just Accepted” Web site may not include all articles that will be publishedin the journal. After a manuscript is technically edited and formatted, it will be removed from the “JustAccepted” Web site and published as an ASAP article. Note that technical editing may introduce minorchanges to the manuscript text and/or graphics which could affect content, and all legal disclaimersand ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errorsor consequences arising from the use of information contained in these “Just Accepted” manuscripts.
1
A Two-Mechanism Model for the Interaction of
Etoposide Quinone with Topoisomerase IIαααα
Elizabeth G. Gibson,†‡ McKenzie M. King,† Susan L. Mercer,†‡ and Joseph E. Deweese†¶*
Department of Pharmaceutical Sciences, Lipscomb University College of Pharmacy and Health Sciences, Nashville, Tennessee 37204-3951 and Departments of Pharmacology and Biochemistry, Vanderbilt University
School of Medicine, Nashville, Tennessee 37232-0146
* To whom correspondence should be addressed: Address: Dept. of Pharmaceutical Science,One University Park Drive, Nashville, TN 37204-3951; Phone: 615-966-7101; Fax: 615-966-7163; E-mail, [email protected]
† Lipscomb University College of Pharmacy and Health Sciences, Department of
Pharmaceutical Sciences. ‡ Vanderbilt University School of Medicine, Department of Pharmacology.
¶ Vanderbilt University School of Medicine, Department of Biochemistry.
Running title:
Etoposide Quinone Uses Two Mechanisms to Impact Topoisomerase IIα
Page 1 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
2
TOC Graphic
Page 2 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
3
ABSTRACT
Topoisomerase II is an essential nuclear enzyme involved in regulating DNA topology to
facilitate replication and cell division. Disruption of topoisomerase II function by
chemotherapeutic agents is in use as an effective strategy to fight cancer. Etoposide is
an anticancer therapeutic that disrupts the catalytic cycle of topoisomerase II and
stabilizes enzyme-bound DNA strand breaks. Etoposide is metabolized into several
species including active quinone and catechol metabolites. Our previous studies have
explored some of the details of how these compounds act against topoisomerase II. In
our present study, we extend those analyses by examining several effects of etoposide
quinone on topoisomerase IIα including whether the quinone impacts ATP hydrolysis,
DNA ligation, cleavage complex persistence, and enzyme:DNA binding. Our results
demonstrate that the quinone inhibits relaxation at 100-fold lower levels of drug when
compared to etoposide. Further, the quinone inhibits ATP hydrolysis by topoisomerase
IIα. The quinone does appear to stabilize single-strand breaks similar to etoposide
suggesting a traditional poisoning mechanism. However, there is minimal difference in
cleavage complex persistence in the presence of etoposide or etoposide quinone. In
contrast to etoposide, we find that etoposide quinone blocks enzyme:DNA binding,
which provides an explanation for previous data showing the ability of the quinone to
inactivate the enzyme over time. Finally, etoposide quinone is able to stabilize the N-
terminal protein clamp implying an interaction between the compound and this portion of
the enzyme. Taken together, the evidence supports a two-mechanism model for the
effect of the quinone on topoisomerase II: 1) interfacial poison and 2) covalent poison
that interacts with the N-terminal clamp and impacts the binding of DNA.
Page 3 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
4
INTRODUCTION
Replication, transcription, and even mitosis are dependent upon regulation of DNA
topology.1-3 This essential task is assigned to a class of enzymes known as DNA
topoisomerases. These enzymes generate transient DNA strand breaks to alleviate
topological impediments. There are two types of topoisomerases: Type I, which create
single-stranded DNA brakes that allow for alleviation of torsional strain, and Type II,
which create double-stranded DNA breaks that facilitate relaxation, unknotting, and
decatenation.1, 2
Mammals have two isoforms of type II topoisomerases: topoisomerase IIα and
IIβ. Topoisomerase IIα (TopoIIα) is up-regulated in response to cell growth and peaks
during mitosis, making it an ideal cancer therapy target.3 TopoIIβ appears to function
during transcription and does not fluctuate as widely through the cell cycle.3 TopoIIα is
the focus of our current study because of its central role as an anti-cancer drug target.
There are broadly two classes of compounds that impact TopoII: catalytic
inhibitors and interfacial poisons.3-5 Inhibitors effect the catalytic cycle of the
topoisomerase enzyme and decrease cleavage complexes leading to slow cell growth
causing quiescence and mitotic failure. Poisons lead to stabilization of TopoII:DNA
complexes (known as cleavage complexes) that results in strand breaks and cell death
or repair of the damage in sub-lethal circumstances. In addition, some compounds
poison TopoII in a non-traditional manner and are known as covalent poisons or redox-
dependent poisons.6 These compounds often share various characteristics including
covalent binding to the enzyme, poisoning of DNA cleavage, and sensitivity to reducing
agents.
Page 4 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
5
Etoposide is an anti-cancer therapeutic that targets TopoII and is used to treat a
variety of cancer types including both solid tumors and hematologic malignancies.4, 5
Etoposide acts as an interfacial poison of TopoII, which lead to strand breaks as noted
above.4, 5, 7 Around 2-3% of patients treated with this agent develop a secondary
leukemia associated with specific chromosomal translocations.8-13 The mechanism for
leukemogenesis resulting from this therapy has not been fully clarified.14
Etoposide is metabolized by CYP3A4 to generate quinone and catechol
metabolites, which may contribute to leukemogenic translocations.15-18 Both of these
metabolites have activity against TopoII.19-21 Previous studies with TopoIIα have shown
that the quinone metabolite displays characteristics of a covalent poison, including 5-
fold higher levels of DNA cleavage and producing a higher ratio of double-stranded to
single-stranded break ratio than etoposide.20 Conversely, the catechol metabolite works
similarly to the parent compound but can also be oxidized to the quinone, which makes
this form less stable and potentially more toxic than etoposide.19, 20 Furthermore,
etoposide quinone induced high levels of DNA cleavage with TopoIIβ at half of the drug
concentration needed with TopoIIα and reacted 2-4 times faster with the β isoform.21
ATP stimulates DNA cleavage with the β isoform in the presence of etoposide but not in
the presence of etoposide quinone.21 The increased activity of the quinone against both
isoforms of TopoII has led us to further explore the differences in the mechanism of
action of etoposide and the quinone metabolite on the TopoIIα isoform.
It is unclear if the quinone only exerts its action using an interfacial poison or if it is
also acting outside of the active site. Using previous data as a guide, we performed
studies to further clarify a hypothesized dual mechanism of the drug working both inside
and outside the active site. Using purified TopoIIα, we investigated the ability of the
Page 5 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
6
quinone to effect DNA relaxation, DNA ligation, stability of cleavage complexes, and the
ATPase activity of the enzyme. Furthermore, we studied the effect of the metabolite on
enzyme:DNA binding by using a mobility shift assay, fluorescence anisotropy, and a
clamp closing assay. Taken together, our data outlined below provide evidence that
etoposide quinone utilizes at least two distinct mechanisms against TopoII: 1) inhibition
of religation (interfacial poisoning) and 2) interaction with the N-terminal clamp
(stabilization of the clamp and blocking of DNA binding). We propose a two-mechanism
model for the action of etoposide quinone.
EXPERIMENTAL PROCEDURES
Enzymes and Materials. Wild-type TopoIIα was expressed in Saccharomyces cerevisiae
JEL1∆top1 cells and purified as described previously.22 The enzyme was stored at -
80ºC as a 1.5 mg/mL (4 µM) stock in 50 mM Tris-HCl, pH 7.7, 0.1 mM EDTA, 750 mM
KCl, 5% glycerol, and 40 µM DTT (carried from the enzyme preparation).
Negatively supercoiled pBR322 DNA was prepared using a Plasmid Mega Kit
(Qiagen) as described by the manufacturer. Etoposide and 1,4-benzoquinone were
obtained from Sigma. Etoposide quinone was synthesized as previously described.20
Drugs were stored at 4°C as 20 mM stock solutions in 100% DMSO, except 1,4-
benzoquinone which was stored as a 20 mM stock in H2O.
Topoisomerase II-mediated Relaxation of Plasmid DNA. Reaction mixtures contained
4.4 nM wild-type human TopoIIα, 5 nM negatively supercoiled pBR322 DNA, and 1 mM
ATP in 20 µL of 10 mM Tris-HCl, pH 7.9, 175 mM KCl, 0.1 mM NaEDTA, 5 mM MgCl2,
and 2.5% glycerol. Assays were started by the addition of enzyme, and DNA relaxation
mixtures were incubated for 15 min at 37°C. DNA relaxation reactions were carried out
in the presence of 0−200 µM etoposide or etoposide quinone. DNA relaxation was
Page 6 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
7
stopped by the addition of 3 µL of stop solution (77.5 mM Na2EDTA, 0.77% SDS).
Samples were mixed with 2 µL of agarose gel loading buffer, heated for 2 min at 45°C,
and subjected to gel electrophoresis in 1% agarose gels. The agarose gel was then
stained in ethidium bromide for 30 min. DNA bands were visualized by UV light and
quantified using a Bio-Rad ChemiDoc MP Imaging System and Image Lab Software
(Hercules, CA). Results were plotted using GraphPad Prism 6 (La Jolla, CA). DNA
relaxation was monitored by the conversion of supercoiled plasmid DNA to relaxed
topoisomers.
Thin-Layer Chromatography-Based ATPase Assay. ATP hydrolysis was monitored
using thin-layer chromatography (TLC) on a polyethylenimine (PEI) matrix (Merck
KGaA, Darmstadt, Germany). Reaction mixtures contained 140 nM of wild-type of
human topoisomerase IIα, 5 nM negatively supercoiled pBR322 DNA, and 1 mM ATP in
20 µL of 10 mM Tris-HCl, pH 7.9, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 2.5%
glycerol. Reactions were incubated at 37oC and 4 µL samples were taken out at
increasing time points (0-30 min) and spotted on the TLC plate. Reactions were run in
the absence (1% DMSO as a control) or presence of etoposide, etoposide quinone, or
etoposide catechol. The plate was then placed in 400 mM ammonium carbonate inside
the TLC chamber and resolved. Separation of ADP from ATP was imaged using an
AlphaImager system (Santa Clara, CA) and quantified using AlphaImager software.
Data were calculated as the percent of ATP converted to ADP in each reaction.
Topoisomerase II-mediated Cleavage of Plasmid DNA. Plasmid DNA cleavage
reactions were performed using the procedure of Fortune and Osheroff.23 Reaction
mixtures contained 220 nM of wild-type human TopoIIα and 5 nM negatively supercoiled
pBR322 DNA in 20 µL of 10 mM Tris-HCl, pH 7.9, 100 mM KCl, 1 mM EDTA, 5 mM
Page 7 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
8
MgCl2, and 2.5% glycerol. Final reaction mixtures contained ~1 µM DTT, which
represents the residual DTT carried along from the enzyme preparation. Unless stated
otherwise, assays were started by the addition of enzyme, and DNA cleavage mixtures
were incubated for 6 min at 37°C. DNA cleavage reactions were carried out in the
absence or presence of 0−200 µM etoposide and etoposide quinone. DNA cleavage
complexes were trapped by the addition of 2 µL of 5% SDS followed by 2 µL of 250 mM
NaEDTA, pH 8.0. Proteinase K was added (2 µL of a 0.8 mg/mL solution), and reaction
mixtures were incubated for 30 min at 37°C to digest TopoIIα. Samples were mixed with
2 µL of agarose gel loading buffer (60% sucrose in 10 mM Tris-HCl, pH 7.9), heated for
2 min at 45°C, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris-
acetate, pH 8.3, and 2 mM EDTA containing 0.5 µg/mL ethidium bromide. Double-
stranded DNA cleavage was monitored by the conversion of negatively supercoiled
plasmid DNA to linear molecules. DNA bands were visualized by UV light and quantified
using a Bio-Rad ChemiDoc MP Imaging System and Image Lab Software (Hercules,
CA). Results were plotted using GraphPad Prism 6 (La Jolla, CA).
Topoisomerase II-mediated Ligation of Plasmid DNA. Ligation assays were performed
using chemical means to induce ligation. DNA cleavage/ligation equilibria were
established with 220 nM wild-type TopoIIα for 6 min at 37°C using the same protocol
above for plasmid-mediated DNA cleavage.
In addition to stopping a control reaction with SDS, ligation reactions were
treated with either 2 µL of 250 mM EDTA or 2 µL of 5 M NaCl prior to 2 µL of 5% SDS.
Addition of EDTA or NaCl to the reaction induces ligation through either metal ion
chelation or changing the ionic strength, respectively. Linear DNA product was used to
quantify double-strand breaks (DSB), while nicked plasmid was used to quantify single-
Page 8 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
9
strand breaks (SSB). Samples were processed, resolved, and analyzed as described
under the plasmid DNA cleavage method above. Results are shown relative to the level
of DNA cleavage in the absence of compound, which was set to a value of 1 (not shown
on figure).
Persistence of Topoisomerase IIα-DNA Cleavage Complexes. The persistence of
TopoIIα-DNA cleavage complexes established in the presence of drugs was determined
using the procedure of Gentry, et al.24 Initial reactions contained 550 nM wild-type
human TopoIIα enzyme, 50 nM DNA, and 25 µM etoposide or 25 µM etoposide quinone
in a total of 20 µL of human cleavage buffer. Reactions were incubated at 37°C for 6
min and then diluted 25-fold with human cleavage buffer warmed to 37°C. Samples (20
µL) were removed at times ranging from 0-240 min, and DNA cleavage was stopped
with 2 µL of 5% SDS followed by 2 µL of 250 mM EDTA (pH 8.0). Samples were
digested with proteinase K and processed as described above for cleavage assays.
Levels of DNA cleavage were set to 100% at time zero, and the persistence of cleavage
complexes was determined by the decay of the linear reaction product over time.
Electrophoretic Mobility Shift Assay to Assess Enzyme:DNA Binding. The ability of DNA
to bind to TopoIIα was measured using an EMSA. Reactions consisting of 0-330 nM
TopoIIα, DNA, 50 mM Tris, pH 7.9, 150 mM KCl, 0.5 mM NaEDTA, and 12.5% glycerol
were incubated at 37°C and carried out in the presence of 10% DMSO or 50 µM
etoposide quinone or 1,4-benzoquinone. Reactions were run: 1) with no drug (DMSO);
2) with enzyme and DNA reacting prior to the addition of compound; or 3) with enzyme
and compound reacting prior to the addition of DNA. Reactions were processed by
adding sample loading buffer and immediately subjected to gel electrophoresis in a 1%
TBE gel stained with ethidium bromide. Gels were imaged using BioRad ChemiDoc MP
Page 9 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
10
Imaging system (Hercules, CA). Binding was qualitatively analyzed by DNA migration
through the gel.
Fluorescence anisotropy to monitor DNA binding to Topoisomerase IIα. A 40-mer
sequence has been labeled on the top strand with 6-FAM (6-carboxyfluorescein) based
upon a previously published sequence.25 Sequences for the strands are as follows: ‘top’
strand, 5’- CGCAATCTGACAATGCGCTCATCGTCATCCTCGCGACGCG-3’ and
‘bottom’ strand, 5’-CGCGTGCCGAGGATGACGATGAGCGCATTGTCAGATTGCG-3’.
Reactions were carried out in an 80 µL reaction mixture with an enzyme titration from 0-
150 nM human TopoIIα, 1 nM DNA, 50 mM KCl, +/-10 mM MgCl2, 50 mM Tris, pH 8.5,
5% glycerol, 10 µg/µL BSA. Reactions were run in the presence of 10% DMSO (no
drug) or 50 µM etoposide or etoposide quinone, which was added to the enzyme and
incubated for 5 minutes at 37°C. Enzyme was titrated into the reaction and successive
fluorescence readings were measured on a Cytation3 imaging plate reader from Bio-
Tek (Winooski, VT) with the appropriate filter sets and anisotropy values were
calculated using BioTek’s Gen5 software. The reactions are run in quadruplicate and
fluorescent anisotropies calculated for each titration point were read ~10 times and
averaged together. Data were analyzed using GraphPad Prism 6 (La Jolla, CA) and
fitted to a one-site specific binding with Hill slope curve. Statistical analysis was
performed within Prism 6 using a one-way ANOVA followed by a Tukey’s Post-Test
Analysis.
Protein N-terminal clamp closing assay. The stabilization of the N-terminal protein
clamp was measured using a modified version of a previously described protocol.26-28
Briefly, 88 nM wild-type human TopoIIα and 2 nM pBR322 were incubated for 10 min at
37°C in a total of 50 µL of clamp closing buffer (50 mM Tris-HCl, pH 8.0, 100 mM KCl, 1
Page 10 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
11
mM EDTA, and 8 mM MgCl2) in the absence or presence of 100 µM etoposide,
etoposide, quinone, or 1,4-benzoquinone. Control reactions including DNA only and
etoposide quinone in the presence of 100 µM dithiothreitol (DTT) were also performed.
After 10 min incubation, 2 mM ATP was added and an addition 10 min incubated was
carried out at 37°C.
Binding mixtures were then loaded into filter baskets containing glass fiber filters
(Millipore) that were pre-equilibrated using clamp closing buffer. Filters were spun at low
speed (~1 krpm) for 5-10 s. Reactions were then washed in 50 µL clamp closing buffer
(low salt), 100 µL of high salt wash (1 M NaCl), and 100 µL of SDS wash (10 mM Tris-
HCl, pH 8.0, 1 mM EDTA, and 0.5% SDS) heated to 65°C. Baskets were transferred to
new tubes after each wash. Eluates were precipitated in isopropanol and dried.
Samples were then resuspended in nucleic acid loading buffer (Bio-Rad) and
electrophoresed in a 1% agarose TAE gel containing ethidium bromide. Gels were
imaged using BioRad ChemiDoc MP Imaging system (Hercules, CA). Supercoiled DNA
bands were quantified for low salt, high salt, and SDS wash eluates for each condition
and DNA eluting after the SDS wash was calculated as a percentage of the total from all
three washes. Data were analyzed used GraphPad Prism 6 (La Jolla, CA), and
statistical analysis was performed using a one-way ANOVA followed by a Tukey’s Post-
Test Analysis.
RESULTS AND DISCUSSION
Etoposide quinone is more potent than etoposide at inhibiting relaxation. As seen
in Figure 1, etoposide and etoposide quinone both inhibit relaxation by TopoIIα.
Page 11 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
12
Interestingly, etoposide quinone inhibits relaxation with ~100-fold less compound than
etoposide (Figure 1). While this complements previous data that shows etoposide
quinone is very potent when studying DNA cleavage20, the current results do not clarify
the mechanism by which relaxation is inhibited. Interfacial poisons, like etoposide, have
the ability to inhibit relaxation through poisoning the DNA cleavage/ligation process.
However, other mechanisms may also impair relaxation, such as inhibition of ATP
hydrolysis by some catalytic inhibitors. The ability of etoposide quinone to impair
relaxation at such low levels could be caused by a different mode of action than the
interfacial poisons or by a combination of mechanisms. We set out to elucidate
alternative mechanisms for etoposide quinone to act upon TopoIIα using a series of
assays.
Etoposide quinone inhibits ATP hydrolysis. Strand passage by TopoIIα is ATP
dependent, and ATP hydrolysis is required for full catalytic activity. Some agents can
block ATP hydrolysis either directly or as a consequence of disrupting the catalytic
cycle. As seen in Figure 2, etoposide has a minor effect on ATP hydrolysis, while
etoposide quinone strongly inhibits hydrolysis by TopoIIα. While this may simply reflect
the ability of this metabolite to poison DNA cleavage and block the enzyme from
ligating, it may also be due to other effects. To further assess ATPase inhibition, we
found that similar to etoposide, etoposide catechol also does not inhibit ATP hydrolysis
at concentrations up to 200 µM (Figure S1). It should be noted that etoposide does
inhibit ATP hydrolysis by yeast TopoII29, but additional analysis indicates that this
inhibition occurs after phosphate release.30 The disparity between our results with
etoposide and TopoIIα and the results with yeast TopoII may reflect fundamental
mechanistic distinctions between the enzymes and/or differences in the techniques
Page 12 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
13
used to measure ATP hydrolysis. Further analysis will be required to clarify this matter.
Therefore, we set out to further explore how etoposide quinone is inhibiting ATP
hydrolysis while also poisoning DNA cleavage.
Etoposide quinone blocks ligation at one scissile bond. Previous results
demonstrated that etoposide quinone does inhibit ligation.20 However, the results were
focused on ligation of double-stranded DNA breaks without examining the single-
stranded DNA breaks. Therefore, we monitored both double- and single-stranded DNA
breaks formed by human TopoIIα under conditions that induce DNA ligation. As seen in
Figure 3, etoposide quinone induces far higher levels of double-stranded DNA breaks
(DSB) and a higher proportion of DSB to single-strand breaks (SSB) when the reactions
are terminated by SDS, which traps the reaction and denatures the enzyme. The
addition of EDTA prior to SDS allows for ligation of cleaved DNA. The results show that
in the presence of EDTA DSBs with both etoposide and etoposide quinone decrease
significantly, while the single-strand breaks increase. Notably, single-strand breaks with
etoposide quinone increase to a significant degree above SSB in reactions terminated
with SDS (~4-fold increase). Further, the addition of NaCl, which promotes dissociation
of the DNA from the enzyme and thereby induces ligation, leads to a decrease in DSBs
and SSBs with both etoposide and the quinone.
Based upon the results discussed above, etoposide quinone inhibits ligation,
similar to etoposide and interfacial poisons. However, this mechanism alone does not
explain the high degree of double-stranded DNA breaks. Therefore, we hypothesized
that a second mechanism may be involved. Since our previous results demonstrate that
the quinone can inactivate DNA cleavage when incubated with the enzyme prior to
Page 13 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
14
DNA20, we used a series of experiments to explore the impact of the compound on the
persistence of enzyme:DNA complexes and on the ability of the enzyme to bind to DNA.
Etoposide and etoposide quinone have a similar effect on the persistence of DNA
cleavage complexes. Since etoposide quinone strongly induces double strand breaks,
the stability of the TopoII:DNA complex in the presence of etoposide quinone was
examined in comparison with complexes formed in the presence of etoposide. In this
experiment, DNA cleavage assays with human TopoIIα were run in the presence of
either etoposide or etoposide quinone and then diluted 10-fold in reaction buffer.
Samples were taken from the diluted reaction over time and stopped using SDS.
Results seen in Figure 4 track the cleavage levels detected over time, which are
indicative of TopoII:DNA complexes. Throughout the four-hour time course, there is no
statistically significant trend or difference between complexes formed in the presence of
etoposide versus those formed in the presence of the quinone. However, it does appear
that complexes with etoposide quinone persist longer than those formed in the presence
of etoposide. This result, however, does not measure whether etoposide quinone can
impede the ability of the enzyme to bind to DNA. Therefore, we set out to examine DNA
binding in the presence of the quinone.
Etoposide quinone impairs DNA binding. As discussed above, etoposide quinone
inhibits the ability of TopoII to ligate cleaved DNA. However, previous studies have also
demonstrated the ability of etoposide quinone to inactivate TopoII activity when the
compound incubated with the enzyme prior to adding DNA.20 While the inhibition of
ligation may be a consequence of a traditional interfacial poisoning mechanism, the
ability to inactivate TopoII activity reflects the mechanism seen with some redox-
dependent or covalent poisons, such as 1,4-benzoquinone.6 We hypothesized that the
Page 14 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
15
ability to inactivate the enzyme may imply the ability of etoposide quinone to block DNA
binding to the TopoII. In order to determine whether etoposide quinone is inhibiting
enzyme:DNA binding, we performed electrophoretic mobility shift assays (EMSA) to
observe the change in migration of DNA in the gel when bound to TopoIIα. As seen in
Figure 5, the covalent poison 1,4-benzoquinone impairs DNA binding when present with
the enzyme prior to the addition of DNA. A similar effect is seen to a lesser extent in the
presence of etoposide quinone, which suggests that the metabolite may reduce
enzyme:DNA binding.
In order to quantitate DNA binding by TopoIIα in the presence of etoposide or
etoposide quinone, we employed fluorescence anisotropy using a fluorescently-labeled,
duplex oligonucleotide. TopoIIα binding decreases the rotation of the DNA substrate in
solution, resulting in higher anisotropy, which is measured as a polarized emission
signal. If etoposide quinone interferes with enzyme:DNA binding, then the anisotropy
will be diminished in the presence of the compound relative to that observed in its
absence.
As seen in Figure 6, increasing concentrations of human TopoIIα bind to the
oligonucleotide resulting in an increasing fluorescence anisotropy signal. The binding
curve is increased in the presence of Mg2+, which is required for DNA cleavage by the
enzyme. The presence of etoposide does not appear to significantly change binding
with or without Mg2+ when compared to the absence of drug. However, etoposide
quinone leads to a 3-4-fold decrease in DNA binding compared to etoposide or the no
drug control, regardless of the presence of Mg2+. The effect is evident when comparing
the calculated Bmax values for each set (Figure 7). The drop in Bmax in the presence of
50 µM etoposide quinone (with Mg2+) is significant (p < 0.05) when compared to no drug
Page 15 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
16
with Mg2+. Comparisons of the Bmax in the absence of Mg2+ shows a change that is
significant at the p < 0.1 level. Based upon binding curve analysis, there is no significant
change in Kd under any of the conditions (Table 1). Therefore, etoposide quinone can
impair binding of DNA to the enzyme when present with the enzyme prior to the addition
of DNA. As discussed below, this result may clarify how etoposide quinone can
inactivate DNA cleavage by the enzyme.
Etoposide quinone stabilizes the N-terminal clamp of TopoIIα. The ability of
etoposide quinone to block binding suggests a structural effect on the enzyme of some
type. Previous research has shown that reactive quinones are able to block the N-
terminal protein clamp of TopoII.28 Therefore, we tested whether etoposide quinone
could stabilize the N-terminal protein clamp using an assay to measure the stability of
the enzyme:DNA complex.26, 27 The protein clamp closing assay examines the stability
of enzyme:DNA complexes by using successive washes of low salt, high salt, and SDS
solutions. Stabilization of the N-terminal clamp is indicated by DNA that is retained in a
glass fiber filter until the SDS wash. It should be noted that this assay does not measure
the enzyme:DNA cleavage complexes. Instead, the complexes that elute are those
where DNA is not cleaved by TopoII.
As seen in figure 8, DNA alone and TopoIIα with DNA do not remain bound at
significant levels to the glass fiber filters. Etoposide appears to cause a low level of DNA
to remain bound, but this is not statistically significant. However, both etoposide quinone
and 1,4-benzoquinone lead to higher levels of DNA in the SDS wash step. Interestingly,
when etoposide quinone is reacted with DTT prior to the addition of enzyme and DNA,
the ability to stabilize the N-terminal clamp is lost, which is consistent with our previous
work on the redox-sensitive nature of the quinone.19, 20 By stabilizing the N-terminal
Page 16 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
17
clamp, ATP hydrolysis is also disrupted,26, 27 which is consistent with the results from
Figure 2. Taken together, etoposide quinone is able to induce the formation of a salt-
stable closed clamp with TopoII on DNA. The ability of etoposide quinone to stabilize
the N-terminal clamp provides an explanation for how this metabolite inhibits ATP
hydrolysis, blocks DNA binding and inactivates the enzyme. Taken together, these data
provide evidence for action of etoposide quinone outside of the active site of TopoIIα.
Conclusions
The TopoII interfacial poison etoposide is metabolized into active species including a
catechol and a quinone. Our previous studies have demonstrated that etoposide
quinone displays characteristics of a redox-dependent covalent poison that reacts with
TopoII. However, the mechanism has not been fully elucidated. Therefore, we set out to
explore the mechanism of action of etoposide quinone against TopoIIα further.
We found that the quinone inhibits plasmid DNA relaxation by TopoIIα at 100-fold
lower concentration when compared to etoposide (1 µM vs 100 µM). While etoposide
quinone does appear to strongly inhibit ATP hydrolysis, this is likely the effect of both
interfacial poisoning and of stabilization of the N-terminal clamp, as discussed below.
Relaxation is inhibited by interfacial poisons, so we examined the ability of the enzyme
to ligate DNA under different conditions in the presence of etoposide and the quinone.
Our results show that in the presence of EDTA, the DSBs formed by TopoIIα in the
presence of the quinone become SSBs. Therefore, the quinone does appear to be
acting similar to the parent compound and is likely blocking ligation on one strand.
When ligation is induced by adding NaCl, both the DSBs and SSBs are decreased,
which may reflect the fact that some of the action of the quinone is non-covalent in
Page 17 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
18
nature. However, the DSBs after NaCl treatment are still four times higher in the
presence of the quinone when compared to etoposide.
Since the DSBs appeared to maintain some stability, we employed a DNA cleavage
persistence assay to study the comparative persistence of DNA cleavage over time. We
found no significant difference in DNA cleavage persistence after a 4 h incubation in a
dilution-based assay. Therefore, if the quinone complexes are more stable under some
reaction conditions, the current assay was unable to detect that stability.
As mentioned above, etoposide quinone displays the ability to inactivate TopoII
activity when present with the enzyme prior to the addition of DNA. This is not seen in
the presence of etoposide.20 We explored whether this result could be due to the ability
of the quinone to inhibit TopoII:DNA binding. We employed EMSA to examine the ability
of the quinone to block enzyme:DNA binding. Our results show that there is a decrease
in binding, but this result was qualitative.
In order to more fully assess the impact of the quinone on enzyme:DNA binding, we
utilized a fluorescence anisotropy assay using a fluorescently-labeled oligonucleotide
similar to previous studies.25, 31 By measuring the change in fluorescence anisotropy in
the presence of increasing concentrations of TopoIIα, we were able to plot the binding of
the enzyme to DNA. Our data shows that etoposide quinone, unlike etoposide, inhibits
the ability of the enzyme to bind to DNA in a quantitative manner. While there is no
change in Kd, there is a decrease in the Bmax at both 10 and 50 µM etoposide quinone.
This is consistent with the quinone making the enzyme less available for binding to
DNA.
Finally, we performed a clamp-closing assay to measure the ability of etoposide
quinone to stabilize the N-terminal protein clamp. Our results show that etoposide
Page 18 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
19
quinone and 1,4-benzoquinone are able to stabilize the closed clamp, while etoposide
or etoposide quinone with DTT are both unable to stabilize the clamp. This result may
provide a mechanism for how etoposide quinone is able to block DNA binding and
inactivate the enzyme when the metabolite is present with TopoII prior to the addition of
DNA. Further, this result may also explain the strong inhibition of ATP hydrolysis by
etoposide quinone. Stabilization of the N-terminal protein clamp is expected to inhibit
ATP hydrolysis by TopoII.26, 27
Taken together, we propose a two-mechanism model for the interaction of
etoposide quinone with TopoII (Figure 9). First, etoposide quinone can act as an
interfacial TopoII poison and inhibit ligation. Second, the quinone appears to be able to
act, outside the active site, in a way that: A) blocks DNA binding when present before
DNA, which inactivates the enzyme and likely involves protomer adduction19, 20, and B)
promotes increased double-stranded DNA breaks and stabilization of the N-terminal
clamp when the DNA is present before the compound. It is possible that some of the
double-strand DNA breaks result from interfacial poisoning, but this mechanism likely
cannot explain the full enhancement seen in the presence of the quinone. We
hypothesize that the closing of the N-terminal clamp may lead to an increase double-
stranded breaks by stabilizing the enzyme on DNA, but further work will be needed to
explore this connection and determine whether this model holds true. Testing this model
will require additional experimentation including the use of an active site mutant to
determine whether some of the effects of etoposide quinone are dependent upon
poisoning of DNA cleavage. It will also be of interest to explore whether these same
effects are seen with TopoIIβ.
Page 19 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
20
The present work has examined the action of etoposide quinone on the function of
TopoIIα and specifically examined the impact on DNA binding and enzyme function. In
summary, etoposide quinone can block enzyme-DNA binding and inactivate the enzyme
when present prior to DNA. When the enzyme binds to DNA, etoposide quinone can
stabilize the enzyme:DNA complex and result in higher levels of double-stranded
breaks.
ASSOCIATED CONTENT
Supporting information
Figure S1 with ATP hydrolysis by TopoIIα in the presence of etoposide catechol and
1,4-benzoquinone and Table S1 with Kd and Bmax Values for Fluorescence Anisotropy in
the Presence of 10 µM Compound are available in Supporting Information. The
Supporting Information is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 615-966-7101; fax: 615-966-7163; E-mail: [email protected].
Funding
This work was funded in part by a New Investigator Award from the American
Association of Colleges of Pharmacy and by support from Lipscomb University College
of Pharmacy and Health Sciences.
Notes
The authors declare no competing financial interest.
Page 20 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
21
ACKNOWLEDGEMENTS
We thank Dr. Anni Andersen for providing the expression vector for His-tagged human
topoisomerase IIα. We would like to thank Dr. Steve Phipps for helpful discussions
regarding statistical analysis. E.G.G. and M.M.K. were participants in the
Pharmaceutical Sciences Summer Research Program of the Lipscomb University
College of Pharmacy and Health Sciences.
ABBREVIATIONS
Bmax, maximum binding; DTT, dithiothreitol; DSB, double-stranded DNA break; EDTA,
ethylenediaminetetracetic acid; EMSA, electrophoretic mobility shift assay; FAU,
fluorescence anisotropy units; Kd, dissociation constant; SC, supercoiled; SSB, single-
stranded DNA break; TopoII, topoisomerase II.
Page 21 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
22
REFERENCES
(1) Wang, J. C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective.
Nat. Rev. Mol. Cell Biol. 3, 430-440.
(2) Bates, A. D., and Maxwell, A. (2005) DNA Topology. Oxford University Press, New
York.
(3) Nitiss, J. L. (2009) DNA topoisomerase II and its growing repertoire of biological
functions. Nat. Rev. Cancer 9, 327-337.
(4) Deweese, J. E., and Osheroff, N. (2009) The DNA cleavage reaction of
topoisomerase II: wolf in sheep's clothing. Nucleic Acids Res. 37, 738-749.
(5) Pommier, Y., Leo, E., Zhang, H., and Marchand, C. (2010) DNA topoisomerases
and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 17, 421-433.
(6) Gibson, E. G., and Deweese, J. E. (2013) Covalent poisons of topoisomerase II.
Curr. Top. Pharm. 17, 1-12.
(7) Wu, C. C., Li, T. K., Farh, L., Lin, L. Y., Lin, T. S., Yu, Y. J., Yen, T. J., Chiang, C.
W., and Chan, N. L. (2011) Structural basis of type II topoisomerase inhibition by
the anticancer drug etoposide. Science 333, 459-462.
(8) Pui, C.-H., Ribeiro, R. C., Hancock, M. L., Rivera, G. K., Evans, W. E., Raimondi,
S. C., Head, D. R., Behm, F. G., Mahmoud, M. H., Sandlund, J. T., and Crist, W.
M. (1991) Acute myeloid leukemia in children treated with epipodophyllotoxins for
acute lymphoblastic leukemia. N. Engl. J. Med. 325, 1682-1687.
(9) Winick, N. J., McKenna, R. W., Shuster, J. J., Schneider, N. R., Borowitz, M. J.,
Bowman, W. P., Jacaruso, D., Kamen, B. A., and Buchanan, G. R. (1993)
Secondary acute myeloid leukemia in children with acute lymphoblastic leukemia
treated with etoposide J. Clin. Oncol. 11, 209-217.
Page 22 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
23
(10) Smith, M. A., Rubinstein, L., and Ungerleider, R. S. (1994) Therapy-related acute
myeloid leukemia following treatment with epipodophyllotoxins: estimating the
risks. Med. Pediatr. Oncol. 23, 86-98.
(11) Relling, M. V., Yanishevski, Y., Nemec, J., Evans, W. E., Boyett, J. M., Behm, F.
G., and Pui, C. H. (1998) Etoposide and antimetabolite pharmacology in patients
who develop secondary acute myeloid leukemia. Leukemia 12, 346-352.
(12) Smith, M. A., Rubinstein, L., Anderson, J. R., Arthur, D., Catalano, P. J., Freidlin,
B., Heyn, R., Khayat, A., Krailo, M., Land, V. J., Miser, J., Shuster, J., and Vena, D.
(1999) Secondary leukemia or myelodysplastic syndrome after treatment with
epipodophyllotoxins. J. Clin. Oncol. 17, 569-577.
(13) Leone, G., Pagano, L., Ben-Yehuda, D., and Voso, M. T. (2007) Therapy-related
leukemia and myelodysplasia: susceptibility and incidence. Haematologica 92,
1389-1398.
(14) Cowell, I. G., and Austin, C. A. (2012) Mechanism of Generation of Therapy
Related Leukemia in Response to Anti-Topoisomerase II Agents. Int. J. Environ.
Res. Public Health 9, 2075-2091.
(15) van Maanen, J. M., de Vries, J., Pappie, D., van den Akker, E., Lafleur, V. M.,
Retel, J., van der Greef, J., and Pinedo, H. M. (1987) Cytochrome P-450-mediated
O-demethylation: a route in the metabolic activation of etoposide (VP-16-213).
Cancer Res. 47, 4658-4662.
(16) Relling, M. V., Evans, R., Dass, C., Desiderio, D. M., and Nemec, J. (1992) Human
cytochrome P450 metabolism of teniposide and etoposide. J. Pharmacol. Exp.
Ther. 261, 491-496.
Page 23 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
24
(17) Relling, M. V., Nemec, J., Schuetz, E. G., Schuetz, J. D., Gonzalez, F. J., and
Korzekwa, K. R. (1994) O-demethylation of epipodophyllotoxins is catalyzed by
human cytochrome P450 3A4. Mol. Pharmacol. 45, 352-358.
(18) Zhuo, X., Zheng, N., Felix, C. A., and Blair, I. A. (2004) Kinetics and regulation of
cytochrome P450-mediated etoposide metabolism. Drug Metab. Dispos. 32, 993-
1000.
(19) Jacob, D. A., Gibson, E. G., Mercer, S. L., and Deweese, J. E. (2013) Etoposide
Catechol Is an Oxidizable Topoisomerase II Poison. Chem. Res. Tox. 26, 1156-
1158.
(20) Jacob, D. A., Mercer, S. L., Osheroff, N., and Deweese, J. E. (2011) Etoposide
quinone is a redox-dependent topoisomerase II poison. Biochemistry 50, 5660-
5667.
(21) Smith, N. A., Byl, J. A., Mercer, S. L., Deweese, J. E., and Osheroff, N. (2014)
Etoposide quinone is a covalent poison of human topoisomerase IIβ. Biochemistry
53, 3229-3236.
(22) Regal, K. M., Mercer, S. L., and Deweese, J. E. (2014) HU-331 is a catalytic
inhibitor of topoisomerase IIα. Chem. Res. Toxicol. 27, 2044-2051.
(23) Fortune, J. M., and Osheroff, N. (1998) Merbarone inhibits the catalytic activity of
human topoisomerase IIα by blocking DNA cleavage. J. Biol. Chem. 273, 17643-
17650.
(24) Gentry, A. C., Pitts, S. L., Jablonsky, M. J., Bailly, C., Graves, D. E., and Osheroff,
N. (2011) Interactions between the etoposide derivative F14512 and human type II
topoisomerases: implications for the C4 spermine moiety in promoting enzyme-
mediated DNA cleavage. Biochemistry 50, 3240-3249.
Page 24 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
25
(25) Gilroy, K. L., and Austin, C. A. (2011) The impact of the C-terminal domain on the
interaction of human DNA topoisomerase II alpha and beta with DNA. PLoS One 6,
e14693.
(26) Roca, J., and Wang, J. C. (1992) The capture of a DNA double helix by an ATP-
dependent protein clamp: a key step in DNA transport by type II DNA
topoisomerases. Cell 71, 833-840.
(27) Roca, J., Ishida, R., Berger, J. M., Andoh, T., and Wang, J. C. (1994) Antitumor
bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in
the form of a closed protein clamp. Proc. Natl. Acad. Sci. U. S. A. 91, 1781-1785.
(28) Bender, R. P., and Osheroff, N. (2007) Mutation of cysteine residue 455 to alanine
in human topoisomerase IIα confers hypersensitivity to quinones: enhancing DNA
scission by closing the N-terminal protein gate. Chem. Res. Toxicol. 20, 975-981.
(29) Morris, S. K., and Lindsley, J. E. (1999) Yeast topoisomerase II is inhibited by
etoposide after hydrolyzing the first ATP and before releasing the second ADP. J.
Biol. Chem. 274, 30690-30696.
(30) Baird, C. L., Harkins, T. T., Morris, S. K., and Lindsley, J. E. (1999) Topoisomerase
II drives DNA transport by hydrolyzing one ATP. Proc. Natl. Acad. Sci. U.S.A. 96,
13685-13690.
(31) Gilroy, K. L., Leontiou, C., Padget, K., Lakey, J. H., and Austin, C. A. (2006)
mAMSA resistant human topoisomerase IIβ mutation G465D has reduced ATP
hydrolysis activity. Nucleic Acids Res. 34, 1597-1607.
Page 25 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
26
Figure Legends:
Figure 1: Etoposide quinone inhibits plasmid DNA relaxation by TopoIIα. Structures of
etoposide (left) and etoposide quinone (right) are shown above an ethidium bromide
stained relaxation gel. Plasmid DNA relaxation by TopoIIα is monitored by gel
electrophoresis in the absence (+TII) or presence of 0.1-100 µM etoposide or etoposide
quinone. Positions of supercoiled (SC) and relaxed (Rel) plasmids are denoted at right.
Supercoiled plasmid DNA standard is at left (DNA). Results are representative of four
independent experiments.
Figure 2: Etoposide quinone inhibits TopoIIα-mediated ATP hydrolysis. TLC-based
ATPase assays were performed with 1% DMSO (ND, black), 25 µM (blue) or 200 µM
(green) etoposide (Etop), and 25 µM etoposide quinone (EQ, red). Time points were
taken at 10, 20, and 30 min and percent ATP converted to ADP was quantified. Error
bars represent the standard deviation of three or more independent experiments.
Figure 3: Etoposide quinone poisons TopoIIα by inhibiting ligation. Both double-strand
and single-strand breaks were tracked during plasmid DNA cleavage reactions with
human TopoIIα in the presence of 50 µM etoposide or etoposide quinone. Reactions
were run for 6 min and then were treated with SDS (to stop the reaction), EDTA (to
induce ligation) then SDS after 5 min, or NaCl (to induce ligation) then SDS after 5 min.
Error bars represent the standard deviation of three or more independent experiments.
Figure 4: Persistence of TopoIIα−DNA cleavage complexes does not vary significantly
in the presence of etoposide or etoposide quinone. Assays were conducted in the
Page 26 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
27
presence of 25 µM etoposide (Etop, blue) or 25 µM etoposide quinone (EQ, red). For
these reactions, DNA cleavage levels at time zero were set to 100% to allow a direct
comparison and plotted on a logarithmic scale. Error bars represent the standard
deviation of at least three independent experiments.
Figure 5: Etoposide quinone impairs binding of human TopoIIα to DNA. TopoIIα at 220
and 330 nM binds to plasmid DNA in the absence of compound (No Cpd) causing a
slower migration of the DNA through a gel compared with the DNA control lane with
plasmid only. In contrast, 50 µM of either 1,4-benzoquinone or etoposide quinone
impede DNA binding. There is a greater effect when the compound is added to the
enzyme prior to DNA (Pre-Cpd) than when the DNA is present before compound (Pre-
DNA). Gels are representative of three independent experiments.
Figure 6: Etopoisde quinone impairs DNA binding by TopoIIα. Incubation of a 40-mer
HEX labeled oligonucleotide duplex with increasing concentrations of human
topoisomerase IIα were performed in the presence or absence of Mg2+ as denoted and
were treated with 10% DMSO (ND), etoposide (Etop), or etoposide quinone (EQ). Left
panel shows compounds at 10 µM, while the right panel shows compounds at 50 µM.
Curves were fit with a Hill slope using Graphpad Prism. Error bars represent the
standard deviation of three independent experiments.
Figure 7: Etoposide quinone reduces the ability of TopoIIα to be available for binding.
Results are shown for 10% DMSO control (ND), etoposide (Etop), and etoposide
quinone (EQ) with or without Mg2+ with compounds at 10 or 50 µM. Statistically
Page 27 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
28
significant difference (p < 0.05) is denoted by ** and represents the comparison of ND
+Mg vs 50 µM EQ +Mg. Bmax is plotted as fluorescence anisotropy units (FAU). Results
are plotted as the mean and SD of Bmax values calculated by Graphpad Prism.
Figure 8: Etoposide quinone stabilizes the N-terminal clamp of TopoIIα similar to
benzoquinone. Gel image shows a representative gel image for low salt (L), high salt
(H), and SDS (S) washes for etoposide (Etop), etoposide quinone (EQ), and
benzoquinone (BQ). Bar graph depicts the percent of plasmid DNA recovered from
glass fiber filters after washing with an SDS solution by using a total DNA flow through
from low salt, high salt, and SDS washes. DNA without enzyme (DNA, grey) and
enzyme without compound (ND, black) are shown along with enzyme plus DNA in the
presence of 100 µM etoposide (Etop, blue), etoposide quinone (EQ, red), benzoquinone
(BQ, orange), or etoposide quinone with 100 µM DTT (EQ+DTT). Reactions were
incubated for 10 min prior to the addition of ATP followed by an additional incubation
before applying samples to the filters. Statistically significant differences (based upon
one-way ANOVA with Tukey’s multiple comparisons post-test) are shown for Etop vs
EQ (**p < 0.05) and for BQ vs EQ (***P < 0.001). Error bars represent the standard
deviation of four or more independent experiments.
Figure 9: Etoposide quinone appears to use a two-mechanism model to impact TopoII.
First, the metabolite acts in the same manner as etoposide by blocking ligation as an
interfacial poison (blue). Second, the quinone appears to also act as a covalent poison
(red) possibly somewhere around the N-terminal clamp (potentially at more than one
site). This mechanism involves covalent adduction of the protomers, which can lead to
Page 28 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
29
several effects: inactivation of the enzyme likely through the blocking of DNA binding to
the enzyme and can also lead to a stabilized closed-clamp form when DNA is present
before the compound adducts. Further, the high proportion of DSB induced by the
quinone may involve the combination of both mechanisms (purple arrows), but is likely
primarily due to the trapping of the cleaved strand of DNA in the closed clamp (larger
purple arrow). However, it must be noted that for there to be interfacial poisoning and
high levels of DSB, the DNA must be present and bound to the enzyme prior to the
quinone. Image generated using Pymol from PDB ID 4GFH.
Page 29 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
30
Figure 1
DNA +TII 0.1 1 10 100 0.1 1 10 100
Etoposide Etoposide Quinone
µM
Rel
SC
Page 30 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
31
Figure 2
0 10 20 30
0
5
10
15
20
25
Time (Min)
ATP Hydrolysis (%)
ND
EQ 25 µM
Etop 200 µM
Etop 25 µM
Page 31 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
32
Figure 3
SDS
EDTANaCl
SDS
EDTANaCl
SDS
EDTANaCl
SDS
EDTANaCl
0
2
4
6
8
10
Relative DNA Cleavage
Etoposide Etoposide Quinone
SSBDSB SSBDSB
50µm 50µm
Page 32 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
33
Figure 4
Page 33 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
34
Figure 5
No Cpd DNA Control
Pre-DNA Pre-Cpd
330 220
1,4-Benzoquinone Etoposide Quinone
330 220 330 220 330 220 330 220 nM (hTIIα)
Pre-DNA Pre-Cpd
Page 34 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
35
Figure 6
Page 35 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
36
Figure 7
ND +Mg
Etop +Mg
EQ +Mg
ND -Mg
Etop -Mg
EQ -Mg
ND +Mg
Etop +Mg
EQ +Mg
ND -Mg
Etop -Mg
EQ -Mg
0
25
50
75
100
125
150
Bmax (FAU)
10 µM 50 µM
**
Page 36 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
37
Figure 8
DNA
ND
Etop
EQ
BQ
EQ +DTT
0
5
10
15
20
Salt Stable DNA (%)
**
***
L H S L H S L H S
Etop EQ BQ
Page 37 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
38
Figure 9
Page 38 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
39
Table 1
Page 39 of 39
ACS Paragon Plus Environment
Chemical Research in Toxicology
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960