Novel xanthone-polyamine conjugates as catalytic inhibitors of human topoisomerase IIα

Article abstract of DOI:10.1016/j.bmcl.2017.09.011

It has been proposed that xanthone derivatives with anticancer potential act as topoisomerase II inhibitors because they interfere with the ability of the enzyme to bind its ATP cofactor. In order to further characterize xanthone mechanism and generate compounds with potential as anticancer drugs, we synthesized a series of derivatives in which position 3 was substituted with different polyamine chains. As determined by DNA relaxation and decatenation assays, the resulting compounds are potent topoisomerase IIα inhibitors. Although xanthone derivatives inhibit topoisomerase IIα-catalyzed ATP hydrolysis, mechanistic studies indicate that they do not act at the ATPase site. Rather, they appear to function by blocking the ability of DNA to stimulate ATP hydrolysis. On the basis of activity, competition, and modeling studies, we propose that xanthones interact with the DNA cleavage/ligation active site of topoisomerase IIα and inhibit the catalytic activity of the enzyme by interfering with the DNA strand passage step.

Full text of DOI:10.1016/j.bmcl.2017.09.011

Accepted Manuscript
Novel xanthone-polyamine conjugates as catalytic inhibitors of human topoi-
somerase IIα
Elirosa Minniti, Jo Ann W. Byl, Laura Riccardi, Claudia Sissi, Michela Rosini,
Marco De Vivo, Anna Minarini, Neil Osheroff
PII:
S0960-894X(17)30893-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.011
BMCL 25275
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 August 2017
31 August 2017
4 September 2017
Please cite this article as: Minniti, E., Byl, J.A.W., Riccardi, L., Sissi, C., Rosini, M., De Vivo, M., Minarini, A.,
Osheroff, N., Novel xanthone-polyamine conjugates as catalytic inhibitors of human topoisomerase IIα, Bioorganic
& Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.09.011
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Novel xanthone-polyamine conjugates as catalytic inhibitors of
human topoisomerase IIα
Elirosa Minnitia,b, Jo Ann W. Bylc, Laura Riccardib, Claudia Sissid, Michela Rosinia,
Marco De Vivob,e, Anna Minarinia,*, Neil Osheroff c,f,g,
*
aDepartment of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via
Belmeloro 6, 40126 Bologna, Italy
bLaboratory of Molecular Modeling and Drug Discovery, Istituto Italiano di Tecnologia, Via Morego
30, 16163 Genova, Italy
cDepartment of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-
0146, USA
dDepartment of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35131
Padova, Italy
eIAS-5/INM-9 Computational Biomedicine, Forschungszentrum Jülich, Wilhelm-Johnen-Straβe, 52428
Jülich, Germany
fDepartment of Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-6307, USA
gVA Tennessee Valley Healthcare System, Nashville, Tennessee 37212 USA
*Corresponding Authors at: Department of Pharmacy and Biotechnology, Alma Mater Studiorum-
University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy (A. Minarini) and Department of
Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA (N.
Osheroff)
E-mail addresses: anna.minarini@unibo.it (A. Minarini), neil.osheroff@vanderbilt.edu (N.
Osheroff).
Keywords: Xanthone derivative; polyamines; DNA topoisomerase IIα; anticancer drug; catalytic
inhibitor; DNA cleavage.

Abstract
It has been proposed that xanthone derivatives with anticancer potential act as topoisomerase II
inhibitors because they interfere with the ability of the enzyme to bind its ATP cofactor. In order to
further characterize xanthone mechanism and generate compounds with potential as anticancer drugs,
we synthesized a series of derivatives in which position 3 was substituted with different polyamine
chains. As determined by DNA relaxation and decatenation assays, the resulting compounds are potent
topoisomerase IIα inhibitors. Although xanthone derivatives inhibit topoisomerase IIα-catalyzed ATP
hydrolysis, mechanistic studies indicate that they do not act at the ATPase site. Rather, they appear to
function by blocking the ability of DNA to stimulate ATP hydrolysis. On the basis of activity,
competition, and modeling studies, we propose that xanthones interact with the DNA cleavage/ligation
active site of topoisomerase IIα and inhibit the catalytic activity of the enzyme by interfering with the
DNA strand passage step.
Graphical Abstract:
Spermidine
Xanthone
2

Xanthones are a class of heterotricyclic planar compounds that were originally identified as
secondary metabolites in plants and microorganisms.1-3 These compounds display a wide range of
biological activities and represent a prominent scaffold for agents with medicinal properties. Several
xanthones currently are under development as anticancer, anti-inflammatory or anti-thrombotic agents.2,
4-11 A number of these compounds display activity against type II topoisomerases.11-16 Furthermore, the
primary cellular target of gambogic acid, a xanthone derivative in human phase II clinical trials as an
anticancer drug, appears to be topoisomerase IIα.12, 13
Human type II topoisomerases play critical roles in a number of nucleic acid processes, including
DNA replication, transcription, recombination, and chromosome segregation.17-22 These enzymes alter
the topological state of the genetic material by transiently cleaving both strands of the double helix and
passing a second double helix through the enzyme-generated DNA gate. This process is dependent on
the presence of an ATP cofactor. ATP binding promotes the DNA strand passage event, while
hydrolysis of the cofactor is required for enzyme turnover. Type II topoisomerases regulate DNA under-
and overwinding, and more importantly, remove tangles and knots from the human genome.17-22
Humans encode two enzyme isoforms, topoisomerase IIα and IIβ. Topoisomerase IIα is essential for the
survival of proliferating cells and is the isoform that is involved in DNA replication and the segregation
of daughter chromosomes. In contrast, topoisomerase IIβ is not essential for cells survival, but is
important for neurological development. Although the precise function of topoisomerase IIβ is not well
defined, it appears to play an important role in the expression of hormonally regulated genes.17-22
Type II topoisomerases are the target for several commonly prescribed anticancer drugs, including
etoposide, doxorubicin and mitoxantrone.17, 18, 22-27 These drugs act by increasing levels of covalent
topoisomerase II-cleaved DNA complexes (i.e., cleavage complexes), which are intermediates in the
catalytic cycle of the enzyme. As result of drug action, the type II enzymes are converted to cellular
3

toxins that fragment the genome. Drugs that act by this mechanism are called topoisomerase II
poisons.17, 18, 22-27
A second group of cytotoxic drugs that includes novobiocin, merbarone, aclarubicin, and ICRF-193
also targets type II topoisomerases. In contrast to the topoisomerase II poisons, these compounds are
catalytic inhibitors that kill cells by robbing them of the essential activities of the type II enzyme.
Catalytic inhibitors can block topoisomerase II activity by a variety of mechanisms, which can have
important consequences for cells. Whereas drugs that block ATP binding (novobiocin), DNA cleavage,
or strand passage (merbarone) inhibit the catalytic reactions of topoisomerase II, those that disrupt
topoisomerase II-DNA binding (aclarubicin) impair both the structural and catalytic roles of the enzyme.
Furthermore, drugs that allow ATP binding but block the hydrolysis of the high-energy cofactor (ICRF-
193), trap the enzyme on the DNA.28-37
Gambogic acid and several other xanthone derivatives are believed to inhibit human topoisomerase
IIα by binding to the ATPase active site of the enzyme and blocking interactions between the enzyme
and the ATP cofactor.12, 15, 16, 38 This hypothesis is based on i) modeling studies that docked compounds
to the ATPase site of topoisomerase IIα, ii) in vitro experiments that demonstrated the inhibition of ATP
hydrolysis by xanthone-based compounds, and iii) surface plasmon resonance studies that suggested that
gambogic acid could bind the ATP domain of the human enzyme.
However, several lines of evidence suggest that the inhibition of topoisomerase IIα by xanthone
derivatives may be more complex. First, all of the ATPase studies reported for xanthone-based
compounds were carried out in the presence of DNA.12, 15, 16 Because the ATPase activity of type II
topoisomerases is stimulated by DNA binding and strand passage,39-41 interfering with DNA interactions
could manifest itself as an indirect inhibition of ATP hydrolysis. Second, many xanthone-based
compounds bind to DNA.13, 16 Thus, they may be able to interact with the DNA cleavage/ligation active
site of type II topoisomerases. Third, some previously described xanthone derivatives display an IC50 for
4

inhibition of ATP hydrolysis that is >10-fold higher than observed for the inhibition of relaxation.15, 16
This makes it unlikely that the loss of overall catalytic activity could have resulted from interference
with ATP interactions. Fourth, some xanthone-based compounds inhibit the DNA relaxation reaction of
topoisomerase I. This is despite the fact that the type I enzyme has no binding site for ATP.42
Therefore, to further examine the mechanism by which xanthones inhibit topoisomerase IIα, we
synthesized a series of new xanthone polyamine conjugates, 2-5, by inserting at the 3 position a side
chain containing different polyamine moieties, including propandiamine (compound 2), butandiamine
(compound 3), spermidine (compound 4), and spermine (compound 5) (Fig. 1). Substitution at the 3
position is favored over the 1 position because of the proximity of the carbonyl.16 These polyamines
were chosen because a previous study found that the presence of a secondary amine group in the side
chain plays an important role in mediating topoisomerase II-drug interactions.43-45 Furthermore, the
addition of a spermine side chain to the core of etoposide (generating F14512) greatly enhanced the
ability of the drug to act as a topoisomerase II poison and to be taken up by cancer cells with active
polyamine transport systems.43-47
O
OH
OH
O
O
OH
(a)
(b)
OH
OH
+
O
O
HO
OH
O
OH
O
(32%)
+
6 (69%)
7
O
O
OH
(c)
X
n
NH2
X
O
NH
n
OH
n=2, X=CH3
1: n=3, X= CH3 (58%)
8: n=2, X= NHBoc (69%)
9: n=3, X= NHBoc (30%)
10: n=2, X= NBoc(CH2)4NHBoc (32%)
11: n=2, X= NBoc(CH2)4NBoc(CH2)3NHBoc (32%)
12: n=2, X= NHBoc
13: n=3, X= NHBoc
14: n=2, X= NBoc(CH2)4NHBoc
15: n=2, X= NBoc(CH2)4NBoc(CH2)3NHBoc
O
O
OH
X
O
NH
n
OH
2: n=2, X= NH2 (36%)*
3: n=3, X= NH2 (45%)**
4: n=2, X=NH(CH2)4NH2 (60%)**
(d)
5: n=2, X=NH(CH2)4NH(CH2)3NH2 (33%)*
Fig. 1. Structures and synthetic pathway of the compounds utilized in this study. Reagents and Conditions:
(a) ZnCl2, POCl3, 70 °C, 3 hours, 69% yield; (b) epichlorohydrin, K2CO3, DMF, 80 °C, 5 hours, mw, 32%
yield; (c) DMF, 50 °C, 26 hours, 32-80% yield; (d) CF3COOH, CH2Cl2, 0 °C, 2 hours or HCl in dioxane, 0
°C, 2-5 hours, 33-60%yield. Boc = (CH3)3COCO. * = hydrochloride salt; ** = trifluoroacetate salt.
5

Compounds 1-5 were synthesized using the generalized scheme shown in Fig. 1. The key
intermediate, 1-hydroxy-3-(oxiran-2-ylmethoxy)-9H-xanthen-9-one (7), was synthesized by an O-
alkylation reaction of compound 6. The synthesis of compound 7 was performed under microwave
irradiation in order to shorten the reaction time. To join the nucleophilic chains to the xanthone core,
intermediate 7 was coupled with butylamine and the N-Boc protected polyamines 12-15 to generate
compounds 1 and 8-11, respectively. In order to synthesize the final compounds 2 and 5 or 3 and 4, tert-
butyloxycarbonyl (Boc) groups were removed with 4 M HCl in dioxane or with trifluoroacetic acid
(TFA) in CH2Cl2, respectively. All of the compounds were synthesized as racemic mixtures. The
detailed syntheses and physical and chemical characterizations of the compounds are described in the
accompanying Supplementary Data.
As a first step toward characterizing the activities of the xanthone derivatives shown in Fig.1 against
human topoisomerase IIα, the effects of compounds 1-6 on enzyme-mediated DNA cleavage were
determined (Fig. 2). Consistent with previous reports,12, 15, 16 none of the compounds displayed a
FII
FIII
FI
TII 6
1 2 3 4 5 Etop
[Compound] (µM)
Fig. 2. Effects of xanthone derivatives on DNA cleavage mediated by topoisomerase IIα. Results for
compounds 1-6 (2.5 µM, blue; 10 µM, red; 50 µM, yellow; 100 µM, green) on the generation of enzyme-
mediated double-stranded DNA breaks are shown. Due to solubility issues, compound 5 was only used up to
10 µM. DNA cleavage in the presence of 100 µM etoposide (purple) is shown for comparison. DNA
cleavage levels were calculated relative to control reactions that contained no drug (TII, orange) and were set
to 1. Error bars represent standard deviations for 2-3 independent experiments. The inset shows an ethidium
bromide-stained gel of a typical DNA cleavage experiment carried out in the presence of 10 µM compounds
1-6 and 100 µM etoposide. The positions of negatively supercoiled (form I, FI), nicked (form II, FII) and
linear (form III, FIII) DNA are indicated.
6

significant ability to enhance DNA cleavage. Thus, these xanthone derivatives do not appear to act
primarily as topoisomerase II poisons.
Next, we examined the abilities of compounds 1-6 to inhibit the overall catalytic activity of
topoisomerase IIα using a DNA relaxation assay (Fig. 3). All of the compounds inhibited DNA
Compound 2
Compound 1
Compound 3
FII
FI
FII
FI
TII 10 25 50 100 200
TII 0.5 1 2.5
5
7.5 10
TII 0.5 1 2.5
5 7.5 10
Compound 4
Compound 5
Compound 6
FII
FI
FII
FI
TII 0.5 1 2.5
5 7.5 10
TII 0.5 1 2.5
5
7.5 10
TII
5
10 25 50 100 200
[Compound] (µM)
Fig. 3. Effects of xanthone derivatives on DNA relaxation catalyzed by topoisomerase IIα. Results for
compounds 1-6 are shown. Assays containing intact negatively supercoiled DNA in the absence of
topoisomerase IIα (DNA) or negatively supercoiled DNA treated with topoisomerase IIα in the absence of
xanthone derivatives (TII) are shown as controls. The positions of negatively supercoiled (form I, FI) and
nicked (form II, FII) DNA are indicated. Gels are representative of 2-4 independent experiments.
relaxation and fell into three groups regarding potency: compounds 2 and 5 displayed an IC50 ≈ 1 µM,
compounds 3 and 4 displayed an IC50 ≈ 2.5-5 µM, and compounds 1 and 6 displayed an IC50 ≈ 100 µM.
From these results, it appears that the presence of the side chain containing a primary and secondary
amine is critical. However, the number of secondary amines in the chain and their distance from the
primary amine appear to be less important. These results notwithstanding, it should be noted that a
previous study reported that compound 1 displayed an IC50 ≈ 9 µM for the inhibition of DNA relaxation
catalyzed by topoisomerase IIα.16 We could not recapitulate this finding in the present study. Given the
low activity of compound 1 in our hands and the fact that the activity of compound 3 was similar to
other derivatives we tested, we focused on compounds 2 and 4-6 for more detailed studies.
The ability of xanthone derivatives to inhibit the catalytic activity of topoisomerase IIα was
confirmed using a decatenation assay (Fig. 4). Although IC50 values were ~2- to 3- fold higher than
7

TII 0.5 1 2.5
5
7.5 10 15 20
TII 0.5
1
2.5 5 7.5 10 15
TII 0.5
1
2.5
5
7.5 10
TII 5 10 25 50 100 200 300
kDNA
kDNA
Nicked
SC
Nicked
SC
120
120
100
120
100
120
100
100
80
60
80
60
80
60
40
20
0
80
60
40
20
0
40
20
0
40
20
0
0
0.5
1
2.5
5
7.5 10 15 20
0
0.5
1
2.5
5
7.5 10 15
0
5
10 25 50 100 200 300
0
0.5
1
2.5
5
7.5 10
[Compound 2] (µM
)
[Compound 4] (µM)
[Compound 6] (µM)
[Compound 5] (µM)
Fig. 4. Effects of xanthone derivatives on DNA decatenation catalyzed by topoisomerase IIα. Results for
compounds 2, 4-6 are shown. Assays containing intact kDNA in the absence of topoisomerase IIα (DNA) or
kDNA treated with topoisomerase IIα in the absence of xanthone derivatives (TII) are shown as controls. The
positions of intact kDNA at the origin (kDNA), decatenated nicked kDNA minicircles, and decatenated
supercoiled (SC) kDNA minicircles are indicated. Gels are representative of 3 independent experiments.
Quantification of results is shown in the bar graphs at the bottom. Levels of decatenation in the absence of
xanthone derivatives was set to 100%. Error bars represent standard deviations for 3 independent experiments.
observed in the relaxation assay, the order of potency remained similar, with compounds 2, 4, 5 being
much more potent than compound 6.
Polyamines have the potential to bind to the double helix and alter topoisomerase II-DNA
interactions in the absence of a specific interaction with the enzyme.48-50 Consequently, is possible that
the xanthone derivatives inhibit the activity of topoisomerase IIα in a bimodal fashion with the aromatic
core of one molecule binding to the protein and the polyamine tail of another acting through a general
effect on the DNA. Therefore, the importance of the linkage between the spermidine/spermine
polyamine tails and the xanthone core (compound 6) in a single molecule was examined.51
Independently, the IC50 values for the inhibition of DNA relaxation by topoisomerase IIα for spermidine
and spermine were >1 mM (data not shown) and that of compound 6 was >100 µM. As seen in Fig. 5, a
1:1 mixture of compound 6 and spermidine or spermine showed no ability to inhibit DNA relaxation at
10 µM. This is compared to compounds 4 and 5 (which are essentially compound 6 coupled to
spermidine and spermine, respectively), that displayed IC50 values <5 µM. Thus, the linkage between the
xanthone core and the polyamine tails is critical for the potent inhibitory activity of these compounds.
8

TII 0.5 1 2.5 5 7.5 10
TII 0.5 1 2.5 5 7.5 10
FII
FI
FII
FI
100
100
6 + Spmd
6 + Spm
80
60
40
20
0
80
60
40
20
0
5
4
0.0 2.5 5.0 7.5 10.0
0.0 2.5 5.0 7.5 10.0
[Compound] (µM)
[Compound] (µM)
Fig. 5. Covalent linkage of the C3 polyamine moiety to the 1,3-dyhydroxy-9h-xanthen-9-one core is critical
for the inhibition of DNA relaxation catalyzed by topoisomerase IIα. The effects of a 1:1 mixture of 1,3-
dyhydroxy-9h-xanthen-9-one (compound 6) + spermidine (left panel) or + spermine (right panel) on the DNA
relaxation activity of human topoisomerase IIα is shown. Assays containing intact negatively supercoiled
DNA in the absence of topoisomerase IIα (DNA) or negatively supercoiled DNA treated with topoisomerase
IIα in the absence of xanthone derivatives (TII) are shown as controls. The positions of DNA markers are as
shown in Figure 3. Gels are representative of 2 independent experiments. Quantification of results is shown in
the graphs at the bottom. Data for compound 6 + spermidine (Spmd, left) and compound 6 + spermine (Spm,
right) are shown as open circles. Control reactions in the presence of compound 4 (left) and 5 (right) are shown
as closed circles. Error bars represent standard error of the mean for 2 independent experiments.
As discussed above, the mechanism by which a compound inhibits the activity of topoisomerase II
can have profound cellular consequences. Thus, it is important to understand which step of the
topoisomerase II catalytic cycle is affected by xanthones. This catalytic cycle can be divided into six
discrete steps: 1) DNA binding, 2) DNA bending, 3) DNA cleavage, 4) strand passage, 5) DNA
religation, 6) release of the DNA substrate and enzyme turnover.17, 18, 52 ATP binding drives the strand
passage step and ATP hydrolysis is required for enzyme turnover and the completion of the catalytic
cycle.17, 18, 20, 21, 32, 53
To determine whether the xanthone derivatives affect topoisomerase II activity at steps up to and
including DNA scission, we reassessed the effects of the compounds 2, 4-6 on enzyme-mediated DNA
cleavage (Fig. 6). Normally, topoisomerase IIα maintains very low levels of cleavage complexes in the
presence of Mg2+, its physiological divalent cation.54, 55 This makes it difficult to determine the ability of
the drug to inhibit this reaction step. Consequently, the experiments shown in Fig. 6 were carried out
9

Fig. 6. Inhibition of topoisomerase IIα-mediated DNA cleavage by xanthone derivatives. Results for
compounds 2, 4-6 are shown. Reactions were carried out in the presence of CaCl2 to enhance baseline levels of
DNA cleavage by the enzyme. Assays containing negatively supercoiled DNA in the absence of
topoisomerase IIα (DNA) or negatively supercoiled DNA treated with topoisomerase IIα in the absence of
xanthone derivatives (TII) are shown as controls. The positions of DNA markers are as shown in Figure 2
inset. Gels are representative of 3 independent experiments. Quantification of results is shown in the graphs at
the bottom. Levels of DNA cleavage in the absence of xanthone derivatives was set to 100%. Error bars
represent standard deviations for 2-3 independent experiments.
in the presence of Ca2+, which raises baseline levels of DNA cleavage ~10- to 15-fold.54, 56 No
substantial inhibition was observed for xanthone concentrations up to 10 µM, the range in which
compounds inhibited overall catalytic activity. This finding strongly suggests that these compounds do
not impair the overall catalytic activity (DNA relaxation or decatenation) of topoisomerase IIα by
inhibiting any of the reaction steps through DNA cleavage. However, this conclusion comes with the
caveat that the DNA cleavage assay utilizes 150 nM enzyme as compared to the relaxation assay, which
uses 3 nM. Thus, it could take higher concentrations of xanthone derivatives to inhibit the catalytic
activity of topoisomerase IIα under conditions of the cleavage assay. Therefore, as a control, the effects
of compounds 2, 4-6 on DNA relaxation catalyzed by 150 nM topoisomerase IIα were determined (Fig.
7). Similar to the results of Fig. 3, compounds 2, 4, and 5 displayed complete (or near complete)
inhibition of enzyme activity by 10 µM. (Note that 10 µM compound 6 displayed no ability to inhibit
DNA relaxation under either assay condition).
10

Compound 2
Compound 4
FII
FI
FII
FI
TII 0.5 1 2.5 5 7.5 10
TII 0.5 1 2.5 5 7.5 10
Compound 6
Compound 5
FII
FI
FII
FI
TII 2.5 5 10 25 50 100 200
TII 0.5 1 2.5 5 7.5 10
[Compound] (µM)
Fig. 7. Effects of xanthone derivatives on DNA relaxation catalyzed by 150 nM topoisomerase IIα. Results for
compounds 2, 4-6 are shown. Assays containing intact negatively supercoiled DNA in the absence of
topoisomerase IIα (DNA) or negatively supercoiled DNA treated with topoisomerase IIα in the absence of
xanthone derivatives (TII) are shown as controls. The positions of DNA markers are as in Figure 3. Gels are
representative of 2-4 independent experiments.
Taken together, the results shown in Figs. 6 and 7 indicate that the xanthone derivatives act by
inhibiting topoisomerase IIα in a reaction step that follows DNA cleavage. Consequently, we examined
the effects of compounds 4, and 5 on DNA strand passage mediated by the enzyme (Fig. 8). A DNA
catenation assay was employed that utilizes a high concentration (150 nM) of topoisomerase IIα and
replaces the ATP cofactor with the non-hydrolyzable analogue, APP(NH)P. Because APP(NH)P cannot
be hydrolyzed by topoisomerase IIα, this assay monitors enzyme activity through the strand passage
step.52, 57 By using a DNA catenation assay, a single catalytic event moves the DNA substrate away from
the bands of relaxed DNA. Relaxed, rather than negatively supercoiled, DNA was used for this assay
because it is a preferred substrate for catenation. Similar to the results in Fig. 7, compounds 4 and 5 all
displayed IC50 values <10 µM. Because these compounds showed little ability to inhibit DNA cleavage
(Fig. 6), the step that immediately precedes the strand passage step, we conclude that the xanthone
derivatives decrease the overall catalytic activity of topoisomerase IIα by inhibiting the ability of the
enzyme to carry out DNA strand passage.
11

TII 1 2.5 5 7.5 10
TII 1 2.5 5 7.5 10
Cat
Rel
Cat
Rel
120
100
80
120
100
80
60
40
20
0
60
40
20
0
0.0 2.5 5.0 7.5 10.0
0.0 2.5 5.0 7.5 10.0
[Compound 4] ( M)
[Compound 5] (µM)
Fig. 8. Effects of xanthone derivatives on DNA strand passage mediated by topoisomerase IIα. Results for
compounds 4 and 5 are shown. Assays monitored the catenation of relaxed DNA in the presence of the non-
hydrolyzable ATP analogue APP(NH)P so that the enzyme could only carry out one round of DNA strand
passage. Assays containing relaxed DNA in the absence of topoisomerase IIα (DNA) or relaxed DNA treated
with topoisomerase IIα in the absence of xanthone derivatives (TII) are shown as controls. The positions of
relaxed DNA (Rel) and catenated DNA at the origin (Cat) are indicated. Gels are representative of at least 3
independent experiments. Quantification of results is shown in the graphs at the bottom. Levels of catenation
in the absence of xanthone derivatives was set to 100%. Error bars represent standard deviations for at least 3
independent experiments.
Xanthones can inhibit the strand passage step in three different ways: they can bind at the ATP
active site and interfere with topoisomerase IIα-ATP interactions, they can bind at the DNA
cleavage/ligation active site and interfere directly with DNA movement, or they may bind outside of
either active site and cause deleterious conformational changes in the enzyme. On the basis of modeling
studies and the ability of compounds to inhibit enzyme-catalyzed ATP hydrolysis, previous studies
suggested that xanthone derivatives acted by inhibiting topoisomerase IIα-ATP interactions.12, 15, 16, 38
Therefore, we assessed the effects of compound 4 on ATP hydrolysis catalyzed by topoisomerase IIα.
As seen in Fig. 9, compound 4 inhibited ATP hydrolysis with an IC50 ≈10 µM. However, as with earlier
studies, ATPase assay mixtures contained DNA. As discussed above (and shown in Fig. 9), rates of ATP
hydrolysis are stimulated ~ 5-fold by the presence of DNA. Thus, decreased rates of ATP hydrolysis
observed in the presence of compound 4 could be due to interactions of the xanthone derivative at either
12

100
80
60
40
20
0
0
10 20 30 40 50
[Compound 4] (µM)
Fig. 9. Effects of compound 4 on ATP hydrolysis catalyzed by topoisomerase IIα. Assays carried out in the
presence or absence of negatively supercoiled DNA are shown as closed or open circles, respectively. ATPase
activity rate in the presence of DNA and in the absence of compound 4 was set to 100%. Error bars represent
standard error of the mean for 2 independent experiments.
the DNA or ATP sites. In order to distinguish between these two possibilities, the effects of compound 4
on ATP hydrolysis were examined in the absence of DNA (Fig. 9). Under this condition, no inhibition
was observed up to 50 µM xanthone. Thus, we conclude that the xanthone-induced decrease in the rate
of ATP hydrolysis in the presence of DNA is not due to a direct inhibition of ATP binding. Rather, it is
observed because xanthone derivatives inhibit the ability of DNA to stimulate the rate of ATP
hydrolysis. Consistent with this conclusion, ATPase rates generated in the presence of supercoiled
plasmid asymptotically approached those seen in the absence of DNA as the concentration of compound
4 increased (Fig. 9).
To further define the site of interaction of xanthone derivatives on topoisomerase IIα, we used a
competition assay to determine whether these compounds could be inhibiting DNA strand passage (and
ATP hydrolysis) by acting at the DNA cleavage/ligation active site. The competition assay determined
the ability of compounds 2, 4 and 5 (which do not inhibit the DNA cleavage step) to block DNA
cleavage enhancement by etoposide. This drug has been shown to bind at the DNA cleavage/ligation
active site of human type II topoisomerases.58 As seen in Fig. 10, all three compounds competed with
13

etoposide, diminishing the ability of the anticancer drug to induce topoisomerase IIα-mediated DNA
scission. These data strongly suggest that the xanthone derivatives
interact in the vicinity of the DNA cleavage/ligation active site of the human type II enzyme, despite the
fact that they have little effect on the cleavage reaction. A similar conclusion has been drawn for the
binding of some quinolone-derivatives that do not enhance DNA cleavage to eukaryotic or prokaryotic
type II topoisomerases.59, 60
Fig. 10. Ability of xanthone derivatives to inhibit the enhancement of topoisomerase IIα-mediated DNA
cleavage by 100 µM etoposide. Results for compounds 2, 4, and 5 (5 µM, blue; 10 µM, red; 25 µM, green; 50
µM, yellow; 100 µM, orange) on the generation of enzyme-mediated double-stranded DNA breaks are shown.
Due to solubility issues, compound 5 was only used up to 10 µM. DNA cleavage in the presence of 100 µM
etoposide (purple) in the absence of xanthone derivatives set to 100%. Error bars represent standard deviations
for 2-3 independent experiments.
Finally, to determine the feasibility of xanthone binding at the cleavage/ligation active site of
topoisomerase IIα, compound 4 was docked into the topoisomerase II cleavage complex by molecular
modeling. Both the R and S enantiomers were used for modeling studies. Fig. 11 shows the results with
the R enantiomer. Our findings suggest that the heterocyclic moiety of the xanthone derivative
preferentially locates in a region similar to that of the 4’-demethylepipodophyllotoxin core of
etoposide.58 The polyamine chain extends toward the DNA major groove, as was observed in previous
computations of F14512 and other polyamine-conjugates.45 Compound 4 has the potential to form
several favorable interactions with both the enzyme and the DNA. The hydroxyl group along the
14

polyamine chain points toward Gln778, while the central nitrogen atom is in close enough proximity to
form hydrogen bonds with the backbone of either Lys814 or Ala816. The terminal amine is stabilized by
the DNA, as it lays in between the phosphates of DNA bases C+3 and A+4.
Fig. 11. Molecular modeling of compound 4. Docking of compound 4 into the DNA cleavage/ligation active
site of topoisomerase IIα (Top). Superposition between etoposide (pdb: 3QX3) and the docking pose of
compound 4 into the topoisomerase II DNA cleavage complex (Bottom).
In conclusion, xanthone derivatives represent a potent class of topoisomerase II inhibitors with
anticancer potential. Previously, these compounds were believed to inhibit enzyme activity by
interfering with the binding of the high-energy ATP cofactor. However, results of the present study
strongly suggest that xanthone-polyamine conjugates act at the DNA cleavage/ligation active site of
human topoisomerase IIα and impair catalytic activity by blocking the DNA strand passage step of the
topoisomerase II catalytic cycle. Having a greater understanding of xanthone mechanism may allow for
future development of more active derivatives that utilize this important medicinal scaffold.
Acknowledgements
The authors declare no competing financial interests. This research was supported by National
Institutes of Health grant GM033944 to N.O., Italian Association for Cancer Research (AIRC) grant
15

IG n. 18883 to M.D.V., and funds from the University of Bologna (RFO) to A.M. We are grateful to
Elizabeth G. Gibson for critical reading of the manuscript.
Supplementary data
Experimental details for the synthesis and characterization of the xanthone derivatives, as well as
sources of materials and methods for biochemical assays and modeling studies, are available in the
accompanying Supplementary Data.
16

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