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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
To link to this article: https://doi.org/10.1080/14756366.2022.2078320
© 2022 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 23 May 2022.
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SHORT COMMUNICATION
From Antarctica to cancer research: a novel human DNA topoisomerase 1B
inhibitor from Antarctic sponge Dendrilla antarctica
Alessio Ottaviani
a
, Joshua Welsch
b
, Keli Agama
c
, Yves Pommier
c
, Alessandro Desideri
a
, Bill J. Baker
b
and
Paola Fiorani
a,d
a
Department of Biology, University of Rome Tor Vergata, Rome, Italy;
b
Department of Chemistry, University of South Florida, Tampa, FL, USA;
c
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA;
d
Institute of Translational
Pharmacology, National Research Council, CNR, Rome, Italy
ABSTRACT
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.
ARTICLE HISTORY
Received 22 March 2022
Revised 3 May 2022
Accepted 8 May 2022
KEYWORDS
Natural product;
topoisomerase; cancer; drug
development
Introduction
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
1
. 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
2
. 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
3–7
. Human
DNA topoisomerase IB (htop1), is a monomeric enzyme that
relaxes supercoiled DNA cutting a single DNA strand through a
concerted mechanism
3,8–10
. After DNA relaxation occurs, the dou-
ble-stranded DNA is restored by reformation of the DNA and the
enzyme is released
11
. This enzyme is the unique target of camp-
thotecin (CPT) and its derivatives
12
, 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
13,14
.
Other compounds, called inhibitors, act by targeting htop1 by
preventing either the enzyme from binding to DNA or by prevent-
ing the cleavage reaction
15,16
. Some of them are natural products
(NPs)
17
such as berberine
18
, benzoxazines
19
and compounds coor-
dinated with metals such as zinc copper and vanadium
20,21
. 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
properties
17,22
.
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
22–24
. Of the eight com-
pounds displaying activity at 200 lM (data not show), membra-
noid G (MG), from Antarctic sponge Dendrilla antarctica
25
,(Figure
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 paola.fiorani@uniroma2.it 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 bjbaker@usf.edu Department of Chemistry, University of South Florida, Tampa,
FL, USA
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 (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2022, VOL. 37, NO. 1, 1404–1410
https://doi.org/10.1080/14756366.2022.2078320
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)
25
. Briefly, the freeze-dried sample of the
Antarctic sponge Dendrilla antarctica was extracted using ACS
grade methylene chloride (CH
2
Cl
2
). 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
diffraction
25
.
Cells and culture conditions
Dulbecco’s modified Eagle’s 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
described
16
. 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 2–9), 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 2–9), 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 1–9), with 100 lM MG in pre-incubation condition (lanes 10–18), and in simultaneous condition (lanes 19–27), 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.
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 1405
final volume of 30 lL containing a buffer composed of 20 mM
Tris-HCl pH 7.5, 0.1 mM EDTA, 10 mM MgCl
2
,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 enzyme’s 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
2
O.
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
26
.
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
27
. 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
2
, 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
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.
EMSA
DNA mobility shift assay was performed by slightly modifying a
previously described procedure
28
. 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
2
to evaluate cell viability as previously
described
16
. 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
2
, 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
2
. 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.
Results
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(B–D)). 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 10–18)) while when MG
was simultaneously incubated to the reaction mixture, the inhib-
ition was reduced (Figure 1(D, lanes 19–27)). 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 1–9)).
1406 A. OTTAVIANI ET AL.
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
100
m
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 6–9)) while in its absence and in pres-
ence of only DMSO (lanes 2–5) 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 DNA–enzyme 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 DNA–enzyme complex slowing down the religation
29
. 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 4–7)). When the protein was incubated with MG alone, the
band of the cleavable complex was not observed (Figure 2(B,
lanes 8–11)), 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 6–9 and B, lanes 7–10)) 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 2–5) and after MG pre-incubation (lanes 6–9). 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
M, lanes 4–7, 10
m
M CPT þMG (0.1, 1.0, 10, and 100
m
M), lanes
8–11, MG (0.1, 1.0, 10, and 100
m
M).
JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 1407
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
M to 100
m
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
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.
Discussion
Nature has been a source of drugs for the treatment of many
human diseases
30
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
p<0.0001.
1408 A. OTTAVIANI ET AL.
Among them, Antartina isolated from the Antarctic plant
Deschampsia antarctica displays antitumor activity
31
,VariolinB,
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
17
.
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
25
. 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
respectively
32
. 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
33
, 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.
Funding
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.
ORCID
Alessandro Desideri http://orcid.org/0000-0003-1541-4217
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