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From Antarctica to cancer research: a novel human DNA topoisomerase 1B inhibitor from Antarctic sponge Dendrilla antarctica


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Nature has been always a great source of possible lead compounds to develop new drugs against several diseases. Here we report the identification of a natural compound, membranoid G, derived from the Antarctic sponge Dendrilla antarctica displaying an in vitro inhibitory activity against human DNA topoisomerase 1B. The experiments indicate that membranoid G, when pre-incubated with the enzyme, strongly and irreversibly inhibits the relaxation of supercoiled DNA. This compound completely inhibits the cleavage step of the enzyme catalytic mechanism by preventing protein binding to the DNA. Membranoid G displays also a cytotoxic effect on tumour cell lines, suggesting its use as a possible lead compound to develop new anticancer drugs. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
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Journal of Enzyme Inhibition and Medicinal Chemistry
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From Antarctica to cancer research: a novel
human DNA topoisomerase 1B inhibitor from
Antarctic sponge Dendrilla antarctica
Alessio Ottaviani, Joshua Welsch, Keli Agama, Yves Pommier, Alessandro
Desideri, Bill J. Baker & Paola Fiorani
To cite this article: Alessio Ottaviani, Joshua Welsch, Keli Agama, Yves Pommier, Alessandro
Desideri, Bill J. Baker & Paola Fiorani (2022) From Antarctica to cancer research: a novel human
DNA topoisomerase 1B inhibitor from Antarctic sponge Dendrilla�antarctica, Journal of Enzyme
Inhibition and Medicinal Chemistry, 37:1, 1404-1410, DOI: 10.1080/14756366.2022.2078320
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© 2022 The Author(s). Published by Informa
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From Antarctica to cancer research: a novel human DNA topoisomerase 1B
inhibitor from Antarctic sponge Dendrilla antarctica
Alessio Ottaviani
, Joshua Welsch
, Keli Agama
, Yves Pommier
, Alessandro Desideri
, Bill J. Baker
Paola Fiorani
Department of Biology, University of Rome Tor Vergata, Rome, Italy;
Department of Chemistry, University of South Florida, Tampa, FL, USA;
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA;
Institute of Translational
Pharmacology, National Research Council, CNR, Rome, Italy
Nature has been always a great source of possible lead compounds to develop new drugs against several
diseases. Here we report the identification of a natural compound, membranoid G, derived from the
Antarctic sponge Dendrilla antarctica displaying an in vitro inhibitory activity against human DNA topo-
isomerase 1B. The experiments indicate that membranoid G, when pre-incubated with the enzyme,
strongly and irreversibly inhibits the relaxation of supercoiled DNA. This compound completely inhibits
the cleavage step of the enzyme catalytic mechanism by preventing protein binding to the DNA.
Membranoid G displays also a cytotoxic effect on tumour cell lines, suggesting its use as a possible lead
compound to develop new anticancer drugs.
Received 22 March 2022
Revised 3 May 2022
Accepted 8 May 2022
Natural product;
topoisomerase; cancer; drug
Despite the advance in clinical research, the fight against cancer
still has a long way to go. Nowadays, new technologies rely pri-
marily on finding targets as tumour specific antigens (TSA) that
are rare and do not always show an expression level sufficient to
make the therapy effective
. On the other hand tumour associated
antigens (TAA) are overexpressed on cancer cells but also present
on normal cells, with the obvious consequence that treatment will
affect normal cells as well
. For this reason, the identification of
drugs with minimal side effects are fundamental. A well character-
ised tumour target is represented by human DNA topoisomerases,
a class of enzymes involved in solving topological DNA problems
that occur during fundamental cellular process such as DNA repli-
cation, transcription, and chromosome segregation
. Human
DNA topoisomerase IB (htop1), is a monomeric enzyme that
relaxes supercoiled DNA cutting a single DNA strand through a
concerted mechanism
. After DNA relaxation occurs, the dou-
ble-stranded DNA is restored by reformation of the DNA and the
enzyme is released
. This enzyme is the unique target of camp-
thotecin (CPT) and its derivatives
, that intercalate in the nicked
DNA preventing the DNA religation step, thus acting as a poison
arresting the DNA replicative process and creating double strand
breaks that, if not repaired, lead cell to death
Other compounds, called inhibitors, act by targeting htop1 by
preventing either the enzyme from binding to DNA or by prevent-
ing the cleavage reaction
. Some of them are natural products
such as berberine
, benzoxazines
and compounds coor-
dinated with metals such as zinc copper and vanadium
. In the
last few years attention has been focussed on characterising NPs
from organisms that live under extreme condition that could per-
mit the development of metabolites with new therapeutic
With this idea in mind we have started screening a library of
60 isolated NPs against htop1, coming from the marine and
Antarctic worlds since these types of environment have selected
organisms adapted to extreme life conditions, producing NPs with
no counterparts in the terrestrial world
. Of the eight com-
pounds displaying activity at 200 lM (data not show), membra-
noid G (MG), from Antarctic sponge Dendrilla antarctica
1(A)), was selected for further characterisation to investigate the
mechanism of inhibition due to its unique scaffold, drug-like prop-
erties, and abundance of available material. This metabolite is able
to inhibit the cleavage reaction of htop1 by binding to the
enzyme and preventing the interaction between the htop1 and
the DNA substrate. This compound has shown a cytotoxic effect
on cancer cell lines having a fast duplication activity, suggesting it
as a possible novel antitumor drug targeting htop1.
Material and methods
Reagents and drugs
Recombinant htop1 protein (Cat. No. ENZ-306) was purchased
from Prospec (Hamada St. 8 Rehovot 7670308 Israel). Topotecan
(Hycamtin) was purchased from GlaxoSmithKline (Brentford,
Middlesex, TW8 9GS, United Kingdom). MTT (, 3-(4,5-Dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and dimethyl sulf-
oxide (DMSO) were purchased from MERCK (Darmstadt, Germany).
DNA Oligonucleotides CL14-FITC (50-GAAAAAAFITCGACTTAG-30)
CONTACT Paola Fiorani Department of Biology, University of Rome Tor Vergata, Rome, 00133 Italy, Institute of Translational
Pharmacology, National Research Council, CNR, Rome, 00133 Italy; Bill J. Baker Department of Chemistry, University of South Florida, Tampa,
ß2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
2022, VOL. 37, NO. 1, 14041410
labelled with fluorescein isothiocyanate (FITC) at its 50end, CP25-P
phosphorylated at its 50end (50-TAAAAATTTTTCTAAGTCTTTTTTC-
30) and R-11 (50- GAAAAAATTTT) were purchased from Eurofins
Genomics (Sportparkstrasse 2, 85560 Ebersberg, Germany).
Membranoid G isolation and characterization
In 2020, Shilling et al. outlined the isolation of aplysulfurin and
subsequent semisynthetic methanolysis reaction that yielded
membranoid G (MG)
. Briefly, the freeze-dried sample of the
Antarctic sponge Dendrilla antarctica was extracted using ACS
grade methylene chloride (CH
). Reverse phase high perform-
ance liquid chromatography (HPLC) yielded aplysulfurin, which
was treated with methanol (MeOH) to produce semisynthetic
membranoids. Purification of the membranoids was performed
using normal phase HPLC resulting in the isolation of 0.7 mg MG
from 24 mg of aplysulfurin. Spectroscopic analysis was achieved
by nuclear magnetic resonance, mass spectrometry and X-ray
Cells and culture conditions
Dulbeccos modified Eagles medium high glucose, RPMI 1640
medium, foetal bovine serum (FBS), L-glutamine, penicillin/strepto-
mycin, were purchased from Euroclone (Pero, Italy). Complete
media (CM) were supplemented with 10% FBS, 2 mM L-glutamine,
0.1 mg/mL streptomycin, and 100 U/mL penicillin. The ovarian can-
cer cell line SKOV-3 was purchased from Cell Biolabs Inc., and
maintained in DMEM-high glucose, CM. Colorectal adenocarcin-
oma cell line, CACO-2, colorectal carcinoma cell line HCT-116 and
melanoma cell SK-MEL-28, were maintained in RPMI 1640 CM.
Non-small-cell lung cancer cell line, A-549, triple negative breast
cancer cell SUM-159 and MDA-MB-231, HER2/c-erb-2 positive
breast cancer cell line SK-BR-3, and adenocarcinoma HeLa cell line
were maintained in DMEM-high glucose, CM. The cells were tested
for mycoplasma using the PCR detection Kit (Euroclone). The cells
were kept in culture for a maximum of eight passages.
Htop1 purification
Htop1 for agarose based assays was purified as previously
. Briefly the enzyme gene sequence was cloned in a
single copy plasmid and transformed in top1 null EKY3 yeast
strain. Transformed cells were grown on SC-Uracil, with 2% dex-
trose and then in SC-Uracil with 2% raffinose until an optical dens-
ity of A600 ¼1. Protein production was induced with 2%
galactose for 6 h. Cells were disrupted using glass beads and the
enzyme isolated by affinity chromatography. In order to test the
integrity of the protein, the fractions were analysed by SDS-PAGE
and immunoblotting.
Dose dependent and time course relaxation assays
The minimal inhibiting dose of MG on htop1 was assessed
through a dose dependent relaxation assay of negatively super-
coiled DNA pBlueScript KSII (-). The reaction was carried out in a
Figure 1. Relaxation of supercoiled DNA. (A) Membranoid G structure. (B) Relaxation of negative supercoiled DNA plasmid by htop1 at increasing MG concentrations
(lanes 29), lane 1, no drug added and lane 10, no protein added. (C) Pre-treatment of htop1 with MG, at increasing concentrations, before addition of supercoiled
DNA plasmid (lanes 29), lane 1, no drug added and lane 10, no protein added (D) Relaxation of negative supercoiled DNA plasmid in a time course experiment with
DMSO (lanes 19), with 100 lM MG in pre-incubation condition (lanes 1018), and in simultaneous condition (lanes 1927), lane 28, no protein added. The reaction
products are resolved on agarose gel and visualised with ethidium bromide. Dimer indicates dimer supercoiled DNA plasmid; SC indicates super-coiled DNA plasmid.
final volume of 30 lL containing a buffer composed of 20 mM
Tris-HCl pH 7.5, 0.1 mM EDTA, 10 mM MgCl
,5lg/mL acetylated
bovine serum albumin hereafter indicated as TOPOmix 1X,
150 mM KCl, 1 U of purified htop1, 0.5 lg pBlueScript and different
concentrations of MG. As positive control the enzyme was incu-
bated with the same amount of DMSO used to dissolved MG. The
reaction was stopped after 1 h incubation at 37 C by adding 0.5%
SDS stop dye. The same procedure was performed for pre-incuba-
tion experiment, but before adding the DNA, htop1 was pre-
treated for 5 min at 37 C with different concentrations of MG. For
time course experiment the mix previously described was incu-
bated with 100 lM of MG and reactions were stopped at different
time points with SDS. In pre-incubation experiments purified
htop1 was incubated with 100 lM of MG for 5 min at 37 C before
adding the supercoiled DNA. All samples were resolved on 1%
agarose gel and in TBE 1 X buffer containing 48 mM Tris, 45.5 mM
boric acid, 1 mM EDTA. The enzymes ability to relax supercoiled
DNA was visualised through a UV transilluminator after a gel stain-
ing in 0.5 lg/mL ethidium bromide and destaining in dH
Cleavage kinetic and htop1-mediated DNA cleavage reactions
In order to analyse the cleavage kinetics CL14-FITC was annealed
to a CP25-P complementary oligonucleotide to produce the cleav-
age substrate, hereafter indicated as suicide substrate (SS). The
cleavage reaction was carried out at different time points in pres-
ence of 100 lM MG by incubating 0.6 pmol of SS with 1.2 pmol
htop1 (Prospec) as elsewhere described with slight modification
After adding the enzyme, aliquots of 30 ll were removed at differ-
ent times and the reactions were stopped by adding 0.5% SDS
(final concentration). In pre-incubation experiment the enzyme
was incubated with MG for 5 min at 25 C prior the addiction of
the SS. After a precipitation with ethanol, samples were resus-
pended in 5 ll of 1 mg/ml of trypsin and incubated at 37 C for
1 h. The samples were analysed by electrophoresis on denaturing
polyacrylamide gel (7 M urea, 20% Acrylamide). For htop1-medi-
ated DNA cleavage reactions a 30-[32P]-labelled 117-bp DNA oligo-
nucleotide was prepared as previously describe
. The
oligonucleotide contains previously identified htop1 cleavage sites
in 161-bp pBluescript SK() phagemid DNA. Approximately 2 nM
radiolabelled DNA substrate was incubated with recombinant
htop1 in 20 ll of reaction buffer [10 mM Tris-HCl (pH 7.5), 50 mM
KCl, 5 mM MgCl
, 0.1 mM EDTA, and 15 lg/mL BSA] at 25 C for
20 min in the presence of various concentrations of membranoid
G, with or without 10
M CPT. The reactions were terminated by
adding SDS (0.5% final concentration) followed by the addition of
two volumes of loading dye (80% formamide, 10 mM sodium
hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1% bro-
mophenol blue). Aliquots of each reaction mixture were subjected
to 20% denaturing PAGE. Gels were dried and visualised using a
phosphoimager and ImageQuant software (Molecular Dynamics).
The percentage of cleaved substrate for fluorescent experiment
was evaluated by densitometry analysis using ImageLab software.
Plots represent the mean of three independent experiments ana-
lysed by a two-way ANOVA test using GraphPad Prism with
mean ± SD values.
p<0.0001 and p<0.001.
DNA mobility shift assay was performed by slightly modifying a
previously described procedure
. Briefly, 0.1 lg of pBlueScript KSII
(-) supercoiled DNA was incubated in 20 lL reaction with 1 X
TOPOmix, 4 U of purified htop1, 15 mM KCl, 1 mM DTT in the
absence or in presence of MG at 37 C for 30 min, or pre-incubat-
ing the enzyme with 400 lM MG for 5 min before adding the
DNA. As positive control htop1 was incubated with 400 lMof
CPT. The samples were immediately analysed on 1% agarose gel
in TBE buffer, both supplemented with 0.5 lg/ml EtBr.
Cell viability assay
Different tumour cell lines were seeded in a 96-well plate for 24 h
at 37 C, 5% CO
to evaluate cell viability as previously
. Cells were treated with different amounts of MG or
Hycamtin (topotecan, positive cytotoxicity control), ranging from
12.5 lM to 100 lM. As a control, the cells were treated with the
same amount of DMSO. The plates were incubated for 48 h at
37 C under 5% CO
, the medium was then removed and replaced
with 200 lL of fresh media supplemented with 0.5 mg/mL of MTT
reagent. Samples were incubated again for 4 h in an incubator at
37 C, 5% CO
. Before measuring the absorbance at 570 nm, the
medium was replaced with 100 lL of DMSO. Statistical analysis
was evaluated by GraphPad Prism using a two-way ANOVA test,
and the EC50 value was calculated by nonlinear regres-
sion analysis.
Membranoid G inhibits the catalytic activity of htop1
The inhibitory effect of MG on htop1 activity was assessed by a
plasmid relaxation assay (Figure 1(BD)). Purified protein was incu-
bated with a supercoiled plasmid in the absence or presence of
an increasing concentration of MG for 1 h. Samples were analysed
by electrophoresis on agarose gel. The results indicate that MG
inhibits the relaxation activity of htop1 in a dose dependent man-
ner and also as a function of the pre-incubation (Figure 1(C,D)). In
fact, simultaneous addition of enzyme, MG and DNA determines
an inhibition of the relaxation activity from 150 lMMG(Figure
1(B, lane 8)) and is maximal at a drug concentration of 200 lM
(Figure 1(B, lane 9)), although a complete inhibition is never
achieved under these conditions. The assay, carried out after pre-
incubating the enzyme with increasing concentrations of MG
before the addition of DNA, shows a greater inhibitory effect on
htop1 activity, with a strong inhibition starting from 80 lM(Figure
1(C, lane 6)). As a control, to ensure that MG does not affect the
electrophoretic mobility of DNA, the substrate has been incubated
in the absence of htop1 and in presence of the compound (Figure
1(B,C, lane 10)). Since MG is dissolved in DMSO, as additional con-
trol, enzyme activity was evaluated in the presence of an identical
amount of DMSO without MG, to show that DMSO does not affect
the relaxation activity of htop1 (Figure 1(B,C, lane 1)).
To further investigate MG behaviour, we carried out two relax-
ation assays as a function of time to understand whether MG
inhibits htop1 catalytic activity in a reversible or irreversible man-
ner, choosing a concentration of 100 lM. The first experiment was
performed pre-incubating the enzyme while the other one by sim-
ultaneously adding the compound. The result confirms that pre
incubation of htop1 with the MG completely inhibited the cata-
lytic activity of enzyme (Figure 1(D, lanes 1018)) while when MG
was simultaneously incubated to the reaction mixture, the inhib-
ition was reduced (Figure 1(D, lanes 1927)). In both cases we
found that MG acted as an irreversible drug, as evidenced by the
fact that the inhibition is constant all over time. The time course
assay was performed in the presence of DMSO, to confirm that
the solvent has no inhibitory effect (Figure 1(D, lanes 19)).
Cleavage assays in the absence and presence of MG
To characterise which step of htop1 catalytic mechanism is affect
by MG, the activity of enzyme was evaluated on a suicide sub-
strate (SS) in absence and presence of MG in a time course experi-
ment, pre-incubating the drug with the protein. The experiment
was done with a fluorescently labelled SS made by the CL14-FITC
annealed to the CP25-P oligonucleotide phosphorylated at the 50
end to produce a duplex with a 50single-strand overhang (Figure
2(A)). This substrate allows the enzyme to generate a suicide prod-
uct since the cleaved AG-30dinucleotide is too short to be reli-
gated, leaving the enzyme covalently attached to the
oligonucleotide 30-end. 1.2 pmol of enzyme, were incubated with
M MG and the reaction was stopped at different time points
from 1 to 15 min, precipitating the samples in 100% ethanol fol-
lowed by digestion with trypsin. The products resolved on a dena-
turing urea polyacrylamide gel indicate that the cleavage is
inhibited (Figure 2(A lanes 69)) while in its absence and in pres-
ence of only DMSO (lanes 25) the enzyme is cutting as indicated
by the plot of the percentage of the cleaved fragment (CL1)
against time (Figure 2(A bottom panel)).
To investigate the effect of MG on the cleavage/religation equi-
librium, the stability of the covalent DNAenzyme complex was
analysed using a double stranded DNA substrate, radiolabelled on
one of the 30ends. When the enzyme was incubated with DMSO
(Figure 2(B, lane 2)) a very small amount of the cleaved DNA
strand was detected at the preferred DNA cleavage site, as
expected and indicated by the arrow. When htop1 was exposed
to CPT, a dramatic increase of the cleaved DNA fragment was
observed (Figure 2(B, lane 3)), indicating that the equilibrium is
shifted towards cleavage, as the drug reversibly binds to the cova-
lent DNAenzyme complex slowing down the religation
. When
the protein was incubated with increasing concentration of MG, in
presence of CPT, the band of the cleaved fragment was still
observed indicating that the enzyme was cleaving the substrate
permitting the CPT to stabilise the cleaved complex (Figure 2(B,
lanes 47)). When the protein was incubated with MG alone, the
band of the cleavable complex was not observed (Figure 2(B,
lanes 811)), indicating that the drug is not inhibiting the religa-
tion. This experiment opens the possibility of two different scen-
arios: MG is unable to induce htop1-mediated DNA cleavage or, as
suggested by the cleavage assay in Figure 2(A), the compound is
inhibiting protein binding.
DNA mobility shift assay
The cleavage inhibition displayed by MG with htop1 (Figure 2(A,
lanes 69 and B, lanes 710)) may be due either to a catalytic
inhibition of the cleavage reaction or to a prevention of htop1
binding to its DNA substrate. In order to clarify this point, a DNA
mobility shift assay was carried out. The results (Figure 3) indicate
that when the enzyme is pre-incubated with MG (lane 3) there is
Figure 2. Cleavage and religation kinetics. (A) Top panel: cleavage reaction as a function of time of the CL14/CP25 SS, depicted on the top of the figure, in presence
of DMSO (lanes 25) and after MG pre-incubation (lanes 69). In lane 1 the protein has not been added. CL1 represents the DNA strand cleaved by the enzymes at
the preferred cleavage site, indicated by an arrow. Bottom panel: percentage of cleaved SS, plotted against time for the reaction with DMSO (circle) and after MG pre-
incubation (triangle). Data shown are means± SD of 3 independent experiments and were analysed by a two-way ANOVA test.
p<0.0001 and p<0.001. (B)
htop1 cleavage assay gel. From left to right: Lane 1, only DNA, lane 2, htop1þDNA, lane 3 CPT 1
M, lanes 47, 10
M CPT þMG (0.1, 1.0, 10, and 100
M), lanes
811, MG (0.1, 1.0, 10, and 100
a partial inhibition of the binding of htop1 to the substrate, as
demonstrated by the presence of both the supercoiled DNA (SC)
and the relaxed DNA (R). On the other hand, only the relaxed
DNA is observed upon a simultaneous addition of the enzyme,
MG and the supercoiled DNA substrate (lane 4) as it is when only
htop1 is added to the substrate (lane 1). In the presence of CPT,
which inhibits the religation activity of htop1 without interfering
with the binding step of htop1 to DNA, a strong band typical of
the cleaved complex was observed (lane 2).
Cell viability assay
MG showed an inhibitory effect on the htop1 relaxation and
cleavage activity, and a cell viability assay was carried out on sev-
eral cancer cell lines to investigate its potential cytotoxic activity.
Cancer cells were treated for 48 h with different concentrations of
MG ranging from 12.5
M to 100
M(Figure 4). Among the tested
cancer cell lines, HeLa, SUM-159, SKBR-3, HCT-116 and A-549 cells
show a significant reduction of viability at 100
M compared to
control cells in presence of DMSO alone, while CACO-2, SKOV-3
and MDA-MB-231 were no affected by MG. All the cells treated
with TPT, that is selectively targeting htop1 and it is routinely
used as positive control, had a strong viability reduction. To better
evaluate cytotoxicity of MG, it was calculated the effective concen-
tration of cell growth inhibition (EC50) relative to control with the
solvent but without the compound. As shown in Table 1,MG
exhibited a low cytotoxic effect for most cancer cell lines, with a
EC50 values ranging from 0.007 mM to 1.5 mM.
Nature has been a source of drugs for the treatment of many
human diseases
and most of the drugs now available come
from NPs. To find new NPs with novel characteristic we decided
to screen, against htopo1, compounds derived from organisms
living in Antarctica. Indeed, there are several NPs obtained from
Antarctic organism that display interesting therapeutic effect.
Figure 3. DNA mobility shift assay. Lane 1, pBlueScript DNA and htop1; lane 2,
pBlueScript DNA, htop1 and CPT; lane3, pBlueScript DNA, htop1 after MG pre-
incubation and lane 4 in simultaneous addition; lane 5, pBlueScript DNA only. SC:
supercoiled DNA; R: relaxed DNA; C: cleavable complex htop1-DNA-drug.
Figure 4. Cell viability assay on cancer cell lines in presence of MG and TPT. Cytotoxicity of MG (black) and TPT (gray) were tested on several cancer cell lines indi-
cated on the top of the histograms using MTT reagent. TPT is the standard positive cytotoxicity control. The reported data represent three independent experiments
with mean ± SD values analysed by a two-way ANOVA test.
control for comparison test, p<0.01, p<0.001 and
Among them, Antartina isolated from the Antarctic plant
Deschampsia antarctica displays antitumor activity
from the Antarctic sponge Kirckpatrickia variolosa has antitumor
and antiviral properties and a subclass of pyrroloiminoquinone
alkaloids extracted from the Antarctic sponge Latrunculia bifor-
mis, exhibits strong antitumor activity against different can-
cer types
In our screening against htopo1 we found interesting inhibitory
activity from MG, a diterpene from the Antarctic sponge Dendrilla
antarctica. This compound has also been reported to have strong
effect on Leishmania donovani infected macrophages, but its
mechanism of action has not been elucidated
. Here we show an
in vitro activity against htop1, an ubiquitous enzyme present also
in bacteria, virus and parasites such as Plasmodium falciparum and
L. donovani, causing malaria and human visceral leishmania
. Our results show that MG, when pre-incubated
with the enzyme inhibits htop1 in an irreversible manner by bind-
ing to DNA. The metabolite has also a cytotoxic effect on cancer
cell lines whose doubling time is below 30 h, as SUM-159, SK-BR-3,
HCT-116 and HeLa cells. A possible hypothesis is that cells having
a fast replication rate requires a high htop1 activity
, thus
explaining the cytotoxicity and the inhibitory activity of MG
against htop1. However, the wide range of EC50 values, for all cell
lines (Table 1) suggest the presence other targets beside htop1.
These high concentrations required to inhibit cell growth could
be explained by the fact that the MG affect multiple targets
reducing its effect on htop1. The reported data show for the first
time, the effect of MG on htop1 and on tumour cells, suggesting
its possible use as a lead compound to develop new anti-
cancer drugs.
Disclosure statement
The authors have no commercial, proprietary, or financial interest
in the products or companies described in this article.
This work and AO were supported by PNRA (The Italian National
Antarctic Research Program) awarded by the Ministry for the
Education, University and Scientific Research (MIUR), grant number
PNRA18_00005-D. Field work in Antarctica was funded by the US
National Science Foundation awards ANT-0838776 and PLR-
1341339 (B.J.B.) from the Antarctic Organisms and
Ecosystems program.
Alessandro Desideri
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MDA-MB-231 52
SKBR-3 54
Colon cancer HCT-116 1500
CACO-2 50
Melanoma SK-MEL-28 1200
Non-small cell lung cancer A-549 53
Ovarian cancer SKOV-3 0.007
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... To analyze the cleavage kinetics, the CL14-FITC oligonucleotide was annealed to a CP25-P complementary strand to assemble both cleavage and religation substrates, hereafter indicated as suicide substrate (SS). The cleavage reaction was performed at different time points with 100 µM of DM, by incubating 0.6 pmol of SS with 1.2 pmol hTop1 (Prospec), as previously described [30]. The reactions were stopped by adding 0.5% SDS. ...
... To characterize which step of the hTop1 catalytic mechanism is affected by DM, the enzyme activity was evaluated on a suicide substrate (SS) in the absence and presence of DM, in a time course experiment, as in [30]. Briefly, the experiment was performed with a fluorescently labelled SS made by the CL14-FITC annealed to the CP25-P oligonucleotide phosphorylated at the 5 end, to produce a duplex with a 5 single-strand overhang ( Figure 3A). ...
Full-text available
Human topoisomerase 1B regulates the topological state of supercoiled DNA enabling all fundamental cell processes. This enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by nicking one DNA strand and forming a transient protein–DNA covalent complex. The interaction of human topoisomerase 1B and dimethylmyricacene, a compound prepared semisynthetically from myricanol extracted from Myrica cerifera root bark, was investigated using enzymatic activity assays and molecular docking procedures. Dimethylmyricacene was shown to inhibit both the cleavage and the religation steps of the enzymatic reaction, and cell viability of A-253, FaDu, MCF-7, HeLa and HCT-116 tumor cell lines.
DNA topoisomerase I was found to be highly abundant in fast-proliferating tumor cells and is a potential target for anticancer therapy. A series of G-quadruplex-containing oligodeoxynucleotides (ODNs) were designed and used as inhibitors of DNA topoisomerase I. It was demonstrated that ODNs with G-quadruplexes can efficiently inhibit the supercoiled DNA relaxation reaction catalyzed by DNA topoisomerase I. Compared with the other conformations, the parallel propeller-type G-quadruplex was the most efficient DNA topoisomerase I inhibitor. Further studies revealed that integrating G-quadruplexes with duplexes to form quadruplex-duplex hybrids could significantly improve the inhibition efficiency. In addition, a circular ODN that consists of a G-quadruplex motif and DNA topoisomerase I binding site was synthesized and used as a DNA topoisomerase I inhibitor. The results showed that the particularly designed circular ODN displayed high inhibitory efficiency on the activity of DNA topoisomerase I with an IC50 value of 54.8 nM. Moreover, the circular ODN exhibited excellent thermal stability and nuclease resistance. Considering the low cytotoxicity of DNA-based biopharmaceuticals, the design strategy and results reported in this study may shed new light on nucleic acid-based DNA topoisomerase I inhibitor construction for potential anticancer drugs.
Full-text available
Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.
Full-text available
Natural products are widely used as source for drugs development. An interesting example is represented by natural drugs developed against human topoisomerase IB, a ubiquitous enzyme involved in many cellular processes where several topological problems occur due the formation of supercoiled DNA. Human topoisomerase IB, involved in the solution of such problems relaxing the DNA cleaving and religating a single DNA strand, represents an important target in anticancer therapy. Several natural compounds inhibiting or poisoning this enzyme are under investigation as possible new drugs. This review summarizes the natural products that target human topoisomerase IB that may be used as the lead compounds to develop new anticancer drugs. Moreover, the natural compounds and their derivatives that are in clinical trial are also commented on.
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The dominant paradigm holds that spontaneous and therapeutically induced anti-tumor responses are mediated mainly by CD8 T cells and directed against tumor-specific antigens (TSAs). The presence of specific TSAs on cancer cells can only be proven by mass spectrometry analyses. Bioinformatic predictions and reverse immunology studies cannot provide this type of conclusive evidence. Most TSAs are coded by unmutated non-canonical transcripts that arise from cancer-specific epigenetic and splicing aberrations. When searching for TSAs, it is therefore important to perform mass spectrometry analyses that interrogate not only the canonical reading frame of annotated exome but all reading frames of the entire translatome. The majority of aberrantly expressed TSAs (aeTSAs) derive from unstable short-lived proteins that are good substrates for direct major histocompatibility complex (MHC) I presentation but poor substrates for cross-presentation. This is an important caveat, because cancer cells are poor antigen-presenting cells, and the immune system, therefore, depends on cross-presentation by dendritic cells (DCs) to detect the presence of TSAs. We, therefore, postulate that, in the untreated host, most aeTSAs are undetected by the immune system. We present evidence suggesting that vaccines inducing direct aeTSA presentation by DCs may represent an attractive strategy for cancer treatment.
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Immune-checkpoint inhibition provides an unmatched level of durable clinical efficacy in various malignancies. Such therapies promote the activation of antigen-specific T cells, although the precise targets of these T cells remain unknown. Exploiting these targets holds great potential to amplify responses to treatment, such as by combining immune-checkpoint inhibition with therapeutic vaccination or other antigen-directed treatments. In this scenario, the pivotal hurdle remains the definition of valid HLA-restricted tumour antigens, which requires several levels of evidence before targets can be established with sufficient confidence. Suitable antigens might include tumour-specific antigens with alternative or wild-type sequences, tumour-associated antigens and cryptic antigens that exceed exome boundaries. Comprehensive antigen classification is required to enable future clinical development and the definition of innovative treatment strategies. Furthermore, clinical development remains challenging with regard to drug manufacturing and regulation, as well as treatment feasibility. Despite these challenges, treatments based on diligently curated antigens combined with a suitable therapeutic platform have the potential to enable optimal antitumour efficacy in patients, either as monotherapies or in combination with other established immunotherapies. In this Review, we summarize the current state-of-the-art approaches for the identification of candidate tumour antigens and provide a structured terminology based on their underlying characteristics.
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DNA topoisomerase I enzymes relieve the torsional strain in DNA; they are essential for fundamental molecular processes such as DNA replication, transcription, recombination, and chromosome condensation; and act by cleaving and then religating DNA strands. Over the past few decades, scientists have focused on the DNA topoisomerases biological functions and established a unique role of Type I DNA topoisomerases in regulating gene expression and DNA chromosome condensation. Moreover, the human enzyme is being investigated as a target for cancer chemotherapy. The active site tyrosine is responsible for initiating two transesterification reactions to cleave and then religate the DNA backbone, allowing the release of superhelical tension. The different steps of the catalytic mechanism are affected by various inhibitors; some of them prevent the interaction between the enzyme and the DNA while others act as poisons, leading to TopI-DNA lesions, breakage of DNA, and eventually cellular death. In this review, our goal is to provide an overview of mechanism of human topoisomerase IB action together with the different types of inhibitors and their effect on the enzyme functionality.
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Background: Camptothecin (CPT) and its derivatives are currently used as second- or third-line treatment for patients with endocrine-resistant breast cancer (BC). These drugs convert nuclear enzyme DNA topoisomerase I (TOP1) to a cell poison with the potential to damage DNA by increasing the half-life of TOP1-DNA cleavage complexes (TOP1cc), ultimately resulting in cell death. In small and non-randomized trials for BC, researchers have observed extensive variation in CPT response rates, ranging from 14 to 64%. This variability may be due to the absence of reliable selective parameters for patient stratification. BC cell lines may serve as feasible models for generation of functional criteria that may be used to predict drug sensitivity for patient stratification and, thus, lead to more appropriate applications of CPT in clinical trials. However, no study published to date has included a comparison of multiple relevant parameters and CPT response across cell lines corresponding to specific BC subtypes. Method: We evaluated the levels and possible associations of seven parameters including the status of the TOP1 gene (i.e. amplification), TOP1 protein expression level, TOP1 activity and CPT susceptibility, activity of the tyrosyl-DNA phosphodiesterase 1 (TDP1), the cellular CPT response and the cellular growth rate across a representative panel of BC cell lines, which exemplifies three major BC subtypes: Luminal, HER2 and TNBC. Results: In all BC cell lines analyzed (without regard to subtype classification), we observed a significant overall correlation between growth rate and CPT response. In cell lines derived from Luminal and HER2 subtypes, we observed a correlation between TOP1 gene copy number, TOP1 activity, and CPT response, although the data were too limited for statistical analyses. In cell lines representing Luminal and TNBC subtypes, we observed a direct correlation between TOP1 protein abundancy and levels of enzymatic activity. In all three subtypes (Luminal, HER2, and TNBC), TOP1 exhibits approximately the same susceptibility to CPT. Of the three subtypes examined, the TNBC-like cell lines exhibited the highest CPT sensitivity and were characterized by the fastest growth rate. This indicates that breast tumors belonging to the TNBC subtype, may benefit from treatment with CPT derivatives. Conclusion: TOP1 activity is not a marker for CPT sensitivity in breast cancer.
Human topoisomerases comprise a family of six enzymes: two type IB (TOP1 and mitochondrial TOP1 (TOP1MT), two type IIA (TOP2A and TOP2B) and two type IA (TOP3A and TOP3B) topoisomerases. In this Review, we discuss their biochemistry and their roles in transcription, DNA replication and chromatin remodelling, and highlight the recent progress made in understanding TOP3A and TOP3B. Because of recent advances in elucidating the high-order organization of the genome through chromatin loops and topologically associating domains (TADs), we integrate the functions of topoisomerases with genome organization. We also discuss the physiological and pathological formation of irreversible topoisomerase cleavage complexes (TOPccs) as they generate topoisomerase DNA–protein crosslinks (TOP-DPCs) coupled with DNA breaks. We discuss the expanding number of redundant pathways that repair TOP-DPCs, and the defects in those pathways, which are increasingly recognized as source of genomic damage leading to neurological diseases and cancer. Topoisomerases have essential roles in transcription, DNA replication, chromatin remodelling and, as recently revealed, 3D genome organization. However, topoisomerases also generate DNA–protein crosslinks coupled with DNA breaks, which are increasingly recognized as a source of disease-causing genomic damage.
From the CH2Cl2 extract of the Antarctic sponge Dendrilla antarctica we found spongian diterpenes, including previously reported aplysulphurin (1), tetrahydroaplysulphurin-1 (2), membranolide (3), and darwinolide (4), utilizing a CH2Cl2/MeOH extraction scheme. However, the extracts also yielded diterpenes bearing one or more methyl acetal functionalities (5-9), two of which are previously unreported, while others are revised here. Further investigation of diterpene reactivity led to additional new metabolites (10-12), which identified them as well as the methyl acetals as artifacts from methanolysis of aplysulphurin. The bioactivity of the methanolysis products, membranoids A-H (5-12), as well as natural products 1-4, were assessed for activity against Leishmania donovani-infected J774A.1 macrophages, revealing insights into their structure/activity relationships. Four diterpenes, tetrahydroaplysulphurin-1 (2) as well as membranoids B (6), D (8), and G (11), displayed low micromolar activity against L. donovani with no discernible cytotoxicity against uninfected J774A.1 cells. Leishmaniasis is a neglected tropical disease that affects one million people every year and can be fatal if left untreated.
Background: The numbers of topoisomerase I targeted drugs on the market are very limited although they are used clinically for treatment of solid tumors. Hence, studies about finding new chemical structures which specifically target topoisomerase I are still remarkable. Objectives: In this present study, we tested previously synthesized 3,4-dihydro-2H-1,4-benzoxazin-3-one derivatives to reveal their human DNA topoisomerase I inhibitory potentials. Methods: We investigated inhibitory activities of 3,4-dihydro-2H-1,4-benzoxazin-3-one derivatives on human topoisomerase I by relaxation assay to clarify inhibition mechanisms of effective derivatives with EMSA and T4 DNA ligase based intercalation assay. With SAR study, it was tried to find out effective groups in the ring system. Results: While 10 compounds showed catalytic inhibitory activity, 8 compounds were found to be potential topoisomerase poisons. 4 of them also exhibited both activities. 2-hydroxy-3,4-dihydro-2H-1,4-benzoxazin-3-one (BONC-001) was the most effective catalytic inhibitor (IC50:8.34 mM) and ethyl 6-chloro-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-acetate (BONC-013) was the strongest potential poison (IC50:0.0006 mM). BONC-013 was much more poisonous than camptothecin (IC50:0.034 mM). Intercalation assay showed that BONC-013 was not an intercalator and BONC-001 most probably prevented enzyme-substrate binding in an unknown way. Another important result of this study was that OH group instead of ethoxycarbonylmethyl group at R position of benzoxazine ring was important for hTopo I catalytic inhibition while the attachment of a methyl group of R1 position at R2 position were play a role for increasing of its poisonous effect. Conclusion: As a result, we presented new DNA topoisomerase I inhibitors which might serve novel constructs for future anticancer agent designs. Graphical abstract.
All organisms including unicellular pathogens compulsorily possess DNA topoisomerases for successful nucleic acid metabolism. But particular subtypes of topoisomerases exist exclusively in prokaryotes and in some unicellular eukaryotes that are absent in higher eukaryotes. Moreover, topoisomerases from pathogenic members of a niche possess some unique molecular architecture and functionalities completely distinct from their non-pathogenic colleagues. The current review will highlight the unique attributes associated with the structures and functions of topoisomerases from the unicellular pathogens with special reference to bacteria and protozoan parasites. It will also summarize the progress made in the domain pertaining to the druggability of these topoisomerases upon which a future platform for therapeutic development can be successfully constructed.