Tài liệu Báo cáo khoa học: Multi-targeted activity of maslinic acid as an antimalarial natural compound pdf

Multi-targeted activity of maslinic acid as an antimalarial
natural compound
Carlos Moneriz
1,4
, Jordi Mestres
2
, Jose
´
M. Bautista
1,3
, Amalia Diez
1,3
and Antonio Puyet
1,3
1 Departamento de Bioquı
´
mica y Biologı
´
a Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain
2 Chemogenomics Laboratory, Research Unit on Biomedical Informatics (GRIB), Institut Municipal d’Investigacio
´
Me
`
dica and
Universitat Pompeu Fabra, Barcelona, Spain
3 Instituto de Investigacio
´
n del Hospital 12 de Octubre, Universidad Complutense de Madrid, Spain
4 Departamento de Bioquı
´
mica, Facultad de Medicina, Universidad de Cartagena, Colombia

Keywords
apicomplexa; merozoite surface protein;
metalloprotease inhibition; PfSUB1;
phospholipase; plasmodium
Correspondence
A. Puyet, Departamento de Bioquı
´
mica y
Biologı
´
a Molecular IV, Facultad de
Veterinaria, Universidad Complutense de
Madrid, E28040 Madrid, Spain
Fax: +34 913 943 824
Tel: +34 913 943 827
E-mail:
(Received 21 April 2011, revised 14 June
2011, accepted 17 June 2011)
doi:10.1111/j.1742-4658.2011.08220.x
Most drugs against malaria that are available or under development target
a single process of the parasite infective cycle, favouring the appearance of
resistant mutants which are easily spread in areas under chemotherapeutic
treatments. Maslinic acid (MA) is a low toxic natural pentacyclic triterpene
for which a wide variety of biological and therapeutic activities have been
reported. Previous work revealed that Plasmodium falciparum erythrocytic
cultures were inhibited by MA, which was able to hinder the maturation
from ring to schizont stage and, as a consequence, prevent the release of
merozoites and the subsequent invasion. We show here that MA effectively
inhibits the proteolytic processing of the merozoite surface protein com-
plex, probably by inhibition of PfSUB1. In addition, MA was also found

to inhibit metalloproteases of the M16 family by a non-chelating mecha-
nism, suggesting the possible hindrance of plasmodial metalloproteases
belonging to that family, such as falcilysin and apicoplast peptide-process-
ing proteases. Finally, in silico target screening was used to search for other
potential binding targets that may have remained undetected. Among the
targets identified, the method recovered two for which experimental activity
could be confirmed, and suggested several putative new targets to which
MA could have affinity. One of these unreported targets, phospholipase
A2, was shown to be partially inhibited by MA. These results suggest that
MA may behave as a multi-targeted drug against the intra-erythrocytic
cycle of Plasmodium, providing a new tool to investigate the synergistic
effect of inhibiting several unrelated processes with a single compound,
a new concept in antimalarial research.
Introduction
As long as effective vaccines against malaria remain
unavailable, the search for new antimalarial drugs is
still required because of the incomplete protection
obtained with the present therapeutic methods and the
emergence of resistant strains in endemic regions. Most
present and prospective drugs against Plasmodium
falciparum, the causative agent of the most virulent
form of human malaria, have been designed to inter-
fere with essential processes at the blood stage of the
parasite [1], which accounts for the main clinical symp-
toms of disease. Despite the wide variety of potential
targets identified in the intra-erythrocytic cycle of
Abbreviations
IC
50
, half maximal inhibitory concentration; MA, maslinic acid; MSP, merozoite surface protein; PLA2, phospholipase A2; RBCs, red blood

cells; SERA, serine repeat antigen.
FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS 2951
P. falciparum [2] only a few drugs have found applica-
tion as therapeutic agents, like those interfering with
hemozoin polymerization in the vacuole (chloroquine,
quinine, mefloquine and other alkaloids), the dihydro-
folate pathway (pyrimethamine, sulphadoxine, progua-
nil), the mitochondrial electron transport chain
(atovaquone) or triggering of oxidative stress (artemis-
in and derivatives, primaquine).
Maslinic acid (MA) is a natural pentacyclic triter-
pene found in the olive fruit [3]. Different activities
have been reported for MA in a variety of biological
systems. In addition to reported antioxidant [4,5], va-
sorelaxation [6] and anti-tumoural [7,8] activities, MA
has been shown to specifically inhibit glycogen phos-
phorylase [9,10] and protein tyrosine phosphatase 1B
[11], exerting anti-diabetic action. Inhibition of HIV
protease has also been reported for MA as well as
other structurally related compounds like ursolic, epi-
pomolic and tormentic acids [12]. MA appears to dis-
play antiparasitic activities in apicomplexa [13], further
demonstrated in Toxoplasma gondii cultures, where the
likely inhibition of proteolytic activity leads to a reduc-
tion in gliding motility and ultra structural alterations
of the parasite [14].
Previous work from our laboratory showed that
MA inhibits the progress of the intra-eythrocytic stages
of P. falciparum, both in vitro [15] and in vivo [16].
Depending on the timing and extent of treatment,

parasites cultured in the presence of MA display
accumulation of ring, trophozoite or schizont intra-
erythrocytic forms. At low MA doses, the inhibition is
reversible, as removal of MA from the cultures relieves
this hindrance, allowing further maturation of the
parasite. The use of parasitostatic drugs has not been
investigated as a possible alternative or complement to
current drug therapies. Parasitostatic drugs may
enhance the host immune response by delaying the
infection progress and thus facilitating the presentation
of plasmodial antigens during the first infective stages,
therefore favouring the development of the acquired
immune response [16–18].
The actual target of MA on P. falciparum remains
to be investigated. MA does not hinder the formation
of hematin [15], discarding a possible interference
with the formation of hemozoin. Among the above
mentioned previously identified biological processes
affected by MA, the inhibition of proteases and ⁄ or
protein tyrosine phosphatases appears, a priori,as
potential targets for this compound in Plasmodium.
While little is known on protein tyrosine phosphatase
activities in P. falciparum, extensive work has been
devoted to finding and using specific inhibitors of Plas-
modium proteases. The main source of amino acids for
plasmodial protein synthesis derives from haemoglobin
degradation in the food vacuole by a process which
involves several proteases: plasmepsins, falcipains and
falcilysins. Plasmepsin II inhibitors have been devel-
oped based on the structure of the available inhibitors

of cathepsin D [19], a lysosomal protease of mamma-
lian cells, and chalcones and phenothiazines have been
assayed as inhibitors of falcipain-2 [20,21]. Inhibition
of the metalloproteases falcilysin and neutral amino-
peptidases acting at the terminal stages of haemoglobin
degradation are also considered as potential antimalar-
ial targets [22,23]. Other proteases related to the matu-
ration of parasites and the invasive process are also
investigated as possible targets for specific antimalarial
drugs: inhibition of merozoite surface protein (MSP1)
processing protease (PfSUB1) has been shown to
reduce erythrocyte invasion [24], while proteases
involved in merozoite egress of the red blood cell, like
the serine repeat antigen (SERA) family also regulated
by PfSUB1 proteolytic activiy [25], have been shown
to be required for the late-stage development of para-
sites [26].
Computational ligand-based approaches to predict
the potential affinity of compounds have been devel-
oped in the last years. These methods allow the virtual
screening of proteins showing the potential to bind a
given compound among large chemical collections.
This methodology was recently applied to analyse the
polypharmacology of drugs [27,28], to design chemical
libraries directed to protein families [29] and to analyse
the chemogenomic space of cardiovascular diseases
[30]. In this report, a comprehensive analysis of poten-
tial inhibitory activities of MA on P. falciparum has
been carried out, focusing first on the putative protease
inhibition. Furthermore a chemogenomic-based screen-

ing using MA as ligand to predict its most probable
targets was performed, searching for enzymatic activi-
ties and protein binding structures which could eventu-
ally reveal new plasmodial target molecules for this
triterpene and novel strategies in malaria therapy.
Results and Discussion
Inhibition of proteases by MA
It has been previously proposed that MA may inhibit
the activity of proteases of T. gondii [14] and HIV
[12,31]. To ascertain the inhibition range of MA on
the different P. falciparum protease classes, in vitro
enzymatic assays were performed encompassing cyste-
ine, aspartic, serine and metalloproteases. The results,
shown in Table 1, indicate that MA is a strong inhibi-
tor of the metalloprotease thermolysin, showing also
Maslinic acid targets on Plasmodium falciparum C. Moneriz et al.
2952 FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS
low half maximal inhibitory concentration values
(IC
50
) for serine and cysteine proteases. Remarkably,
no inhibition was observed on the aspartic protease
pepsin. However, a possible specific inhibition of plas-
modial aspartic proteases could not be discarded, as
strong inhibition by MA on the HIV protease, which
belongs to the aspartic protease catalytic class, was
previously reported [12]. Accordingly, an additional
inhibition assay was performed using P. falciparum
protein extracts and including cathepsin D (an aspartic
protease) and the aspartic protease inhibitor pepstatin

A as controls. The results (Table 2) showed that MA
does not inhibit cathepsin D nor the aspartic protease
activity in plasmodial protein extracts. A limited inhi-
bition by MA was observed in extracts obtained from
leukocytes. In contrast, the protease inhibitor pepstatin
A showed strong inhibitory activity on all samples.
These results can be explained assuming that MA may
behave as a specific inhibitor of HIV protease, or the
protease class A2 to which the HIV protease belongs,
showing no activity on class A1 proteases (pepsin,
cathepsin D). Remarkably, the only aspartic proteases
predicted from comparative genomic analysis in P. fal-
ciparum belong to the A1 class [32], thus explaining
the lack of inhibition observed in the protein extracts.
These results support the data reported on T. gondii
infections [14], and suggest that the parasitostatic effect
of MA on P. falciparum infected erythrocytes may be
mediated by the inhibition of one or more proteases,
probably corresponding to metalloproteases, serine
and ⁄ or cysteine proteases, which are required to reach
the schizont stage.
The proteolytic hydrolysis of haemoglobin as a
source of amino acids constitutes one of the essential
processes which take place along the intra-erythrocytic
stage of the parasite. Degradation of haemoglobin is
performed in the food vacuole by the combined action
of aspartic proteases (plasmepsins I, II, IV and histoas-
partic protease), cysteine proteinases (falcipains) and a
metalloprotease (falcilysin) [33]. The resulting small
peptides are reduced to dipeptides by aminopeptidases

[23] which may be further hydrolysed to free amino
acids outside the digestive vacuole [34,35]. It has been
shown that cysteine protease inhibitors, such as vinyl
sulfones, reduce the initial cleavage of globin peptides
in the trophozoite vacuole [36,37]. This effect has been
explained either by the direct inhibition of falcipain
[38] or by the indirect effect on the functionality of the
vacuole as a result of the accumulation of partially
hydrolysed peptides, leading to the accumulation of
uncleaved globin [39]. The possible effect, either direct
or indirect, of MA on globin hydrolysis was tested by
incubating synchronized ring-stage parasites with MA
or leupeptin, a cysteine protease inhibitor, and visuali-
zation of the globin band by SDS ⁄ PAGE. The results
did not show the characteristic accumulation of globin
in MA-treated cultures (Fig. 1), indicating that the ini-
tial hydrolysis of haemoglobin is not inhibited by MA.
In addition, the morphology of infected erythrocytes
incubated in the presence of MA is visibly different
from leupeptin-treated cultures. As can be seen in
Fig. 1B, the food vacuoles of parasites incubated 24 h
with leupeptin were abnormally dark-stained due to
the blockage in globin hydrolysis, while MA-treated
cultures showed abnormal trophozoite morphology
due to the growth arrest, but no accumulation of glo-
bin or vacuolization. These results confirm that MA
does not hinder the initial processing of globin and, in
consequence, it is unlikely that falcipains are targeted
by the drug.
Effect of MA on the activity of P. falciparum MSP

As shown in Table 1, MA inhibits subtilisin with an
IC
50
in the range of 50 lm. Subtilisin is a serine prote-
ase of the S8 family closely related to the subtilases 1
(PfSUB1), 2 (PfSUB2) and 3 (PfSUB3) reported in
P. falciparum [32,40]. These proteins play an essential
Table 1. Effect of MA on different representative proteases. All
tests were performed at a fixed concentration of enzyme and
selected according to the detection limit. The MA concentration
range was 1–400 l
M.
Enzyme
Protease
class
Enzyme
(mUÆmL
)1
)
Detection
limit
(mUÆmL
)1
)IC
50
MA (lM)
Subtilisin A Serine 0.4 0.001 59.3 ± 9.2
Thermolysin Metallo 400 1.5 8 ± 3.4
Papain Cysteine 20 0.08 107 ± 12.5
Pepsin Aspartic 5000 0.25 No inhibition

Table 2. Effect of MA on aspartic protease activity in P. falciparum
protein extracts. MA and pepstatin A were tested at 300 l
M.
Enzyme or total
protein extract Compound % Inhibition
Cathepsin D None 0
Pepstatin A 100
MA 6
Parasite None 0
Pepstatin A 100
Schizont MA 0
Leukocyte None 0
Pepstatin A 100
MA 15
C. Moneriz et al. Maslinic acid targets on Plasmodium falciparum
FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS 2953
role in the erythrocyte invasion by the parasite mero-
zoite through a mechanism involving the discharge of
PfSUB1 into the parasitophorous vacuole and the pro-
teolytic activation of SERA proteases, which are
required for merozoite egress [41,42]. An additional
role in the maturation of MSPs (MSP1, MSP6 and
MSP7) has also been recently reported for PfSUB1
[24]. MSPs are involved in the merozoite invasion of
erythrocytes [43]. PfSUB1 function is complemented
by the reported activity of PfSUB2, which performs a
secondary extracellular processing step on the MSP
complex [44]. Due to their similarity with subtilisin,
these subtilases may be expected to be inhibited by
MA. To verify this hypothesis, parasite proteins were

analysed by western blot with mouse anti-P. falciparum
MSP1. Incubation of synchronized cultures at schizont
stage with MA for 12 h led to the inhibition of MSP1
processing, revealed by the detection of the 195 kDa
band, which was not detectable in untreated cultures
at the same cycle time (Fig. 2A). Furthermore, the
morphology of 12 h cultures treated with MA showed
a delay in the maturation of schizonts, which can be
associated with the inhibition of the MSP1 processing
(Fig. 2B). These results indicate that at least PfSUB1
is inhibited by MA in erythrocyte cultures, corroborat-
ing that this compound behaves as a serine protease
inhibitor. Remarkably, the only specific inhibitor of
PfSUB1 reported before is also a natural product,
MRT12113 [24] (Fig. 2C), showing few structural simi-
larities with MA. A comparative study on both mole-
cules might help in the design of simpler structures
behaving as specific inhibitors of this protease.
Noteworthy, it is well established that antibodies
against different regions of the MSP1 complex are
present in populations showing a level of immunity to
P. falciparum malaria [45], and Plasmodium yoelii poly-
morphic variant MSP7-3 has been used to immunize
mice against blood stage infection [46]. The immuniza-
tion of ICR mice treated with MA after a primary
infection observed in our laboratory [16] could then be
related to the inhibition of MSP processing reported
here, as the prolonged exposure of unprocessed MSP
complex would allow the selection of specific neutraliz-
ing antibodies able to bind to released merozoites, thus

hindering invasion of new red blood cells (RBCs).
Chelation-independent protease inhibition by MA
As shown in Table 1, MA is a potent inhibitor of
thermolysin, a bacterial zinc metalloprotease belonging
to the M16 family [47]. Non-specific inhibition of
metalloprotease activity can readily be achieved by
chelating agents that bind to metal cations required in
the active site of the enzyme. The observed inhibition of
MA on thermolysin and PfSUB1, a calcium-dependent
serine protease, might then also be explained if MA
behaves as chelating agent on divalent cations. To test
this possibility, a colorimetric chelation assay was car-
ried out using zinc as divalent cation. As shown in the
Fig. 3, no significant chelation capacity was detected
for MA, even at higher concentrations than those used
in the treatments. This result shows that MA inhibits
metalloprotease and PfSUB1 activities by a specific,
non-chelating mechanism and also reinforces the
250
148
98
64
50
36
22
16
6
4
kDa
MW

1
Leupeptin (24 h)
Start rings (0 h)
Untreated (24 h)
Maslinic acid (24 h)
23 4
AB
Fig. 1. MA-treated cultures do not accumulate undegraded haemoglobin. Synchronized ring-stage P. falciparum was cultured in the pres-
ence of 100 l
M MA or 100 lM leupeptin for 18 h, followed by extraction of proteins and parasite visualization in thin blood smears. (A) Coo-
massie Blue stained 15% SDS ⁄ PAGE of total protein from infected RBC after 18 h of culture, which corresponds to the trophozoite stage.
Lane 1, untreated control parasites; lane 2, culture incubated with leupeptin; lane 3, parasites incubated with MA; lane 4, human haemoglo-
bin standard. 14 kDa bands correspond to undegraded globin monomers. Parasites incubated with the cysteine proteinase inhibitor leupeptin
accumulated undegraded globin, while no differences with the untreated control were observed in MA-treated cultures. (B) Morphological
changes in infected RBC in drug-treated cultures: aliquots of the cultures described above before and after the addition of inhibitor were
obtained at 18 h and stained with Wright’s. The food vacuoles of parasites incubated with leupeptin were abnormally dark-staining due to a
block in globin hydrolysis. Control parasites matured to trophozoite state. Parasites incubated with MA generated abnormal trophozoites, but
no accumulation of haemoglobin was observed.
Maslinic acid targets on Plasmodium falciparum C. Moneriz et al.
2954 FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS
possible inhibition exerted by MA on plasmodial key
metalloprotease activities required for the maturation
of the trophozoite. MA might also specifically inhibit
metalloproteases of the thermolysin class M16. Several
candidates of this family, playing important roles in the
parasite erythrocytic cycle, have been identified by data
mining of P. falciparum proteome [32]: falcilysin, in its
dual role as haemoglobin peptidase and transit peptide
processing activity in the apicoplast [48]; the mito-
chondrial processing peptidases (PFE1155c, PFI1625c),

or insulysin and pitrilysin [32], possibly involved in the
processing of apicoplast protein leader sequence.
Inhibition of any of these activities could contribute to
MA interference in the maturation of the parasite.
Polypharmacology of MA
The observed inhibition on PfSUB1 may contribute to
the arrest of P. falciparum infective cycle detected in
MA-treated cultures [15]. However, the morphology of
the blocked parasite cannot be completely explained
by inhibition of MSP1 processing. MSP1 is synthesized
from the onset of schizogony and is processed by
PfSUB1 at the time of merozoite egress from the
infected erythrocyte. It has been previously shown that
incubation of synchronic cultures with a highly specific
inhibitor of PfSUB1 produced no apparent effect on
pre-schizont stages, but rather a very specific inhibition
of schizont rupture and reduced invasion of the
released merozoites, which can be revealed by accu-
mulation of merozoite parasites in the cultures [41].
In contrast, cultures treated with MA display an
increased fraction of ring, trophozoite or schizont
stages [15], suggesting an additional inhibitory effect
early in the intra-erythrocytic cycle. The probable inhi-
bition of plasmodial metalloproteases by MA opens up
the possibility of a multi-targeted drug, interfering with
different parasite processes and leading to a blockage
of parasite maturation in the RBC from early ring to
schizont stages.
To further investigate the extent of possible multi-
targeted inhibitory activities of MA, a computational

screening was carried out to identify potential binding
targets for MA. Here, in silico target screening was
used to test MA against ligand-based models derived
for 4500 proteins. Table 3 compiles the list of six pro-
teins identified by this method as putative targets for
MA. Two of the proteins retrieved correspond to
previously reported targets for MA, namely protein
tyrosine phosphatase and glycogen phosphorilase,
providing support for the validity of the approach.
To the best of our knowledge, the other four proteins
have never been suggested as possible targets for MA.
Nevertheless, there is compelling evidence of the
connection between these targets and malaria. Phos-
pholipase A2 (PLA2) was detected in P. falciparum-
infected human erythrocytes and found to be inhibited
by the antimalarial drugs chloroquine, quinine and
No drug (12 h)
MA (12 h)
207
78
53
35
28
19
6
114
A
Control
(0 h)
B

Control
(12 h)
MA
(12 h)
Start schizonts (0 h)
kDa
O
O
O
HO
OH
HO
OH
OH
OH
HO
OH
OH
OH
HO
HO
CH
3
H
3
C
CH
3
CH
3

CH
3
H
3
C CH
3
COOH
H
H
H
C
Maslinic acid
MTR12113
Fig. 2. Inhibition of MSP1 primary processing by MA. Highly synchronized early schizonts of P. falciparum 3D7 ( 36 h post-invasion) cul-
tured for 12 h in the presence of MA (100 l
M) were used to obtain an extract of parasite proteins and Wright’s stained thin smears for mon-
itorization of the infective stage. (A) Western blot of the protein extract using the MSP1 antibody as probe. The unprocessed MSP1
( 195 kDa) remains present in cultures treated with MA, while no detectable band was observed in the untreated control. (B) Morphology
of the infected erythrocytes before MA incubation (0 h) and after 12 h incubation in the presence or absence of MA 100 l
M. A delay in the
maturation of schizonts is observed, which may be related to inhibition of MSP1 processing. (C) Structures of MA and the reported PfSUB1
inhibitor MTR12113.
C. Moneriz et al. Maslinic acid targets on Plasmodium falciparum
FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS 2955
artetether at concentrations that cause 50% inhibition
at millimolar concentrations of those drugs [49].
Although the other three possible targets may not be
involved in the effect of MA on the intra-erythrocytic
cycle, they could be relevant to other aspects of
malaria therapy. The polymorphic cytochrome P450

(CYP) isoform 2C8 has been reported to be actively
involved in drug efficacy due to its capacity to metab-
olize antimalarial drugs in humans [50]. On the other
hand, selective and irreversible inhibitors of mosquito
acetylcholinesterases for controlling malaria and other
mosquito-borne diseases have recently been described
[51]. Finally, even though the serotonin 5-HT2B
receptor subtype has not yet been specifically related
to malaria, there are reports linking serotonin recep-
tors in general as potential targets mediating differen-
tial chemical phenotypes in P. falciparum [52]. Given
the high levels of cross-pharmacology among amine
G-protein-coupled receptors [29], if some of them
have been linked already to malaria, the remaining
members of this subfamily could be relevant to
malaria as well.
Among the four novel putative targets identified,
MA was tested on PLA2, since it is the target showing
the highest predicted affinity. The results obtained are
collected in Table 4. As can be observed, MA inhibits
PLA2 in a dose-dependent manner up to 25% at
400 lm, which may be comparable to the 50% inhibi-
tion of PLA2 reported for other antimalarial drugs at
millimolar concentrations [49]. Accordingly, PLA2
may indeed be considered a new target for MA. The
inhibition of plasmodial PLA2, although incomplete,
can be related to the lipid metabolism and membrane
dynamics, contributing to the overall effect of this
compound on parasite maturation when combined
with the observed PfSUB1 and metalloprotease inhibi-

tion.
It is worth stressing that in silico target screening of
MA did not point to any protease or peptidase as
potential target for MA. Considering the increasing
body of evidence of protease inhibition activity of
MA, gathered in this and previous reports, the fact
that MA was found outside the current chemical cov-
erage for those targets may suggest a non-standard
binding mechanism to these proteins. Most natural or
designed protease inhibitors mimic the structure of a
flexible peptidic molecule to bind to the active site.
This approach is based on the observation that prote-
ases frequently bind their inhibitors ⁄ substrates in
extended or b-strand conformation, requiring a linear
arrangement of the substrate backbone [53]. The pen-
tacyclic triterpene structure of MA (Fig. 2C), however,
does not fit with that principle and may represent a
new kind of protease-binding molecule displaying
novel specific inhibition activities. Two lines of evi-
dence support such a target-specific notion: the
reported inhibition of HIV protease [12,54] which is
not extended to other aspartic proteases, and the fact
that MA is present in a variety of food products,
showing no toxic effects by inhibition of human prote-
ases. Should these arguments be confirmed experimen-
tally, MA would be a valuable lead molecule
for development of specific drugs against apicomplexa
parasites.
Concentration (mM)
20 100 200 300

% zinc chelation
0
20
40
60
80
100
50
Fig. 3. Zinc chelating assay for MA. Percentage of zinc chelation
detected using Eriochrome Black T as an indicator of non-com-
plexed zinc cations. The assay was carried out by adding different
amounts of MA (black bars) or EDTA (grey bars) to a solution con-
taining 32 l
M Zn
2+
. Results are expressed as 100 · A
610 nm
(sam-
ple) ⁄ A
610 nm
(control without zinc).
Table 3. Results of the chemogenomic screening of proteins with
high probability of binding to MA.
Enzyme
MA predicted
affinity (l
M)
Phospholipase A2 3.2
Protein tyrosine phosphatase 4.0
CYP2C8 6.3

Acetylcholinesterase 10
5-HT2B 13
Glycogen phosphorylase 40
Table 4. Inhibition of PLA2 activity by MA. MA and 3,4-dichloroiso-
coumarin (DIC) were tested at 100 and 400 l
M. The compounds
were incubated with the enzyme control and schizonts protein for
15 min and 2 h respectively. Results are expressed as the percent-
age of inhibition compared with the control with no inhibitor added.
Enzyme or total
protein extract Inhibitor
Inhibition at
100 l
M (%)
Inhibition at
400 lM (%)
PLA2 bee venom MA 16 25
DIC 18 20
Schizont MA 12 15
DIC 13 18
Maslinic acid targets on Plasmodium falciparum C. Moneriz et al.
2956 FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS
Materials and methods
Drugs and inhibitors
MA was kindly provided by Dr Garcı
´
a-Granados from the
University of Granada, Spain. Leupeptin, pepstatin A and
3,4-dichloroisocoumarin were purchased from Sigma-
Aldrich (St Louis, MO, USA). All drugs were dissolved in

100% dimethylsulfoxide prior to assay.
In vitro cultures of P. falciparum
P. falciparum strain 3D7 (clone MRA-102) was provided
by The Malaria Research and Reference Reagent
Resource Center (MR4, deposited by DJ Carucci). Ery-
throcytes (RBC) were obtained from type A+ healthy
human local donors and collected in tubes with citrate-
phosphate-dextrose anticoagulant (Vacuette
Ò
Greiner Bio-
One GmbH, Kremsmu
¨
nster, Austria). The culture med-
ium consisted of standard RPMI 1640 (Sigma-Aldrich)
supplemented with 0.5% Albumax I (Life Technologies,
Paisley, UK), 100 lm hypoxanthine (Sigma-Aldrich),
25 mm HEPES (Sigma-Aldrich), 12.5 lgÆmL
)1
gentamicine
(Sigma-Aldrich) and 25 mm NaHCO
3
(Sigma-Aldrich).
Each culture was started by mixing uninfected and
infected erythrocytes to achieve a 1% haematocrit and
incubated in 5% CO
2
at 37 °C in tissue culture flasks
(Iwaki Asahi Glass, Tokyo, Japan). The progress of
growth in the culture was determined by microscopy in
thin blood smears stained with Wright’s eosin methylene

blue solution (Merck, Darmstadt, Germany), using the
freely available plasmoscore software [56] to monitor the
parasitaemia. A detailed description of the culture and
synchronization methods used has been published previ-
ously [57].
P. falciparum protein extracts
Proteins from parasite extracts were obtained from a 25-mL
culture of infected RBCs. The cultures were harvested
and the cells resuspended in 1 volume of saponin 0.2%
in NaCl ⁄ P
i
(PBS) 1· and vortexed gently for 5 s to lyse
the RBC membranes. The released parasites were pelleted
at 10 000 g for 10 min and washed three times in cold
PBS. The pellets were solubilized in 50 mm Tris ⁄ HCl pH
8, 50 mm NaCl, 3% Chaps, 0.5% MEGA 10 and gently
mixed at 4 °C for 15 min followed by three freeze–thaw
cycles. After centrifugation (13 000 g, 10 min, 4 °C) the
supernatant was collected and referred to as para-
site extract. Approximately 10 lg of total protein
supernatant was boiled in electrophoresis sample buffer
[5% (wt ⁄ vol) SDS, 62.5 mm Tris ⁄ HCI, 5% (vol ⁄ vol)
2-mercaptoethanol, pH 6.8] and separated on 15%
SDS ⁄ PAGE.
Protease inhibition assays
The protease activities were conducted in vitro using the
internally quenched fluorogenic peptide substrate (EnzChek
Ò
Protease Assay Kit-E33758; Invitrogen). 50 lL of different
protease dilutions, with or without MA at different concen-

trations, were put into separate wells of a microplate. After
the addition of 50 lL of substrate working solution (1.57 mg
in 50% dimethylsulfoxide, 10 mm Tris ⁄ HCl, pH 7.8) the
plate was incubated at room temperature, protected from
light, for 60 min. The fluorescence intensity was measured at
485 nm excitation and 528 nm emission using a Perkin Elmer
LS-50B luminescence spectrophotometer. Background fluo-
rescence was subtracted from the inset data. Plots of percent-
age control activity versus concentration of inhibitor were
used to determine the concentrations that inhibited 50% of
protease activity. The enzymes evaluated, all purchased from
Sigma-Aldrich, were as follows: subtilisin A (
EC 3.4.21.62.)
from Bacillus licheniformis (serine protease), papain (
EC
3.4.22.2) from Carica papaya (cysteine protease), pepsin (EC
3.4.23.1) from porcine gastric mucosa (aspartic protease) and
thermolysin (
EC 3.4.24.27) from Bacillus thermoproteolyti-
cus rokko (metalloprotease). Assays were performed in
10 mm Tris ⁄ HCl (pH 7.8) except for pepsin, which was
assayed in 10 mm HCl (pH 1.8). Dilutions of papain were
made from a buffer containing 30 mml-cysteine.
The effect of MA on the activity of aspartic proteases
present in parasite protein extracts was assayed spectroflu-
orometrically with the internally quenched fluorescent
substrate MCA-G-K-P-I-L-F-F-R-L-K(DNP)-D-Arg-NH
2
trifluoroacetate salt (Sigma-Aldrich), which is not initially
fluorescent due to quenching of the 7-methoxycoumarin-4-

acetyl (MCA) group by the 2,4-dintrophenyl group (DNP).
For the above, 15 lL of protein extract or the control
enzyme (cathepsin D aspartic protease from bovine spleen
provided by Sigma-Aldrich) were disposed in a 96-well mi-
croplate, with or without MA (10 ll). 20 lL of reaction
buffer (0.5 m sodium acetate at pH 5.0) were added and the
volume completed to 98 lL with distilled water. All sam-
ples were incubated at 37 °C for 10 min to allow the inhibi-
tion of the enzyme. After this time, 2 lL of the substrate
were added and the samples were incubated at 37 ° C for
30 min. Parasite protein extracts were also incubated with
the aspartic protease inhibitor pepstatin A as a control.
Finally, the fluorescence signal was measured at 323 nm
excitation and 398 nm emission using a Perkin Elmer LS-
50B luminescence spectrophotometer.
Plasmodial MSP processing assay
To analyse the possible inhibitory activity of MA on the pro-
cessing of the MSP1, synchronous cultures of 3D7 schizonts
(10% parasitaemia) were supplemented with either MA
(100 lm) or dimethylsulfoxide only and cultured for 8–12 h
C. Moneriz et al. Maslinic acid targets on Plasmodium falciparum
FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS 2957
to allow schizont rupture and merozoite invasion. The para-
site pellet was resuspended in PBS containing a complete
Protease Inhibitor Cocktail (Roche, Basel, Switzerland).
After incubation on ice for 10 min, proteins from parasite
extracts were obtained as described above. Parasite proteins
were analysed by western blot with mouse anti-P. falciparum
MSP1 (AbD Serotec, Oxford, UK), followed by horseradish
peroxide conjugated anti-mouse IgG (GE Healthcare, Wau-

keha, WI, USA) at a 1 : 5000 dilution [24]. Detection was
performed using the Super Signal chemiluminescent substrate
(Thermo Fisher Scientific, Rockford, IL, USA) and exposure
to X-ray film. Finally, aliquots from cultures grown were also
examined microscopically.
Haemoglobin degradation assay
To assess the effect of MA on the haemoglobin accumula-
tion in trophozoites, synchronized ring-stage parasites at
10% parasitaemia were incubated at 37 °C in microtitre
plate cultures with MA (100 lm) or the cysteine proteinase
inhibitor leupeptin (100 lm) as a control. After 18 h of
incubation, Wright-stained smears were prepared from cul-
tures, and the parasites were evaluated for the presence of a
marked food vacuole abnormality that has been correlated
with a block in haemoglobin degradation [37,58]. To assess
haemoglobin accumulation, parasites cultured with inhibi-
tors as indicated were collected after 18 h incubation, and
proteins from parasite extracts were obtained as described
above, solubilized in electrophoresis sample buffer and elec-
trophoresed through 15% SDS ⁄ PAGE [36,37]. Proteins
were identified by staining the gel with Coomassie Blue.
In silico target screening
Our in silico target screening approach relies on the assump-
tion that the set of bioactive ligands collected for a given tar-
get provides a complementary description of the target from
a ligand perspective. In order to be able to process this infor-
mation efficiently, chemical structures need to be encoded
using some sort of mathematical descriptors. In this work,
three types of two-dimensional descriptors were used, namely
phrag, fpd and shed, each one of them characterizing chem-

ical structures with a different degree of fuzziness and thus
complementing each other in terms of structural congenerici-
ty and hopping abilities [59]. Then, the probability of any
molecule interacting with a particular target is assumed to be
related to the degree of similarity relative to the set of known
bioactive ligands for that target. A detailed description of the
methodology used can be found elsewhere [60].
Phospholipase inhibition assays
The effect of MA on PLA2 (EC 3.1.1.4) activity was
determined by using a secretory PLA2 assay kit (Cayman
Chemical, Ann Arbor, MI, USA) on 96-well microplates.
Each well contained 10 lL of dinitrobenzoic acid (10 mm
3,5-dinitrobenzoic acid in 0.4 m Tris ⁄ HCl, pH 8.0), 10 l Lof
parasite protein extract or 10 lL of PLA2 bee venom sup-
plied by the kit as positive control, and 5 lL of the corre-
sponding inhibitor: MA or 3,4-dichloroisocoumarin
dissolved in dimethylsulfoxide. The reactions were initiated
by adding 200 lL substrate solution [1.66 mm 1,2-bis(hepta-
noylthio)glycerophosphocholine] to all the wells and the
plate was shaken carefully. Absorbances were monitored at
414 nm using a plate reader every minute for controls and
every hour for parasite samples. Absorbance data were
expressed as a percentage of inhibitory activity compared
with control without inhibitor. Assays were performed in
25 mm Tris ⁄ HCl, pH 7.5, containing 10 mm CaCl
2
, 100 mm
KCl and 0.3 mm Triton X-100.
Chelating activity assay
The metal chelating activity was measured using the com-

plexometric indicator Eriochrome Black T (Sigma-Aldrich).
Samples were prepared by mixing 100 l L of 100 l m
ZnSO
4
.H
2
O, 200 lL of 0.3 m Na
2
CO
3
pH 10, 10 lL of com-
pound (MA or EDTA as control) and 6 lL of Eriochrome
Black T (5 mgÆmL
)1
in ethanol, pH 10). The pH of the Erio-
chrome Black T solution was adjusted by adding buffer solu-
tion dropwise until the colour changed from purple to blue.
200 lL of each sample were transferred to a 96-well micro-
plate and the absorbance was measured at 610 nm. Data
were expressed as a percentage of the increase in absorbance
caused by the removal of zinc cations due to chelating activ-
ity compared with a control without zinc. EDTA was used as
a positive control for chelating activity.
Acknowledgements
This work was supported by grants from the Spanish
Ministry of Education and Science (BIO2007-67885
and BIO2010-17039) and the Research Teams Consoli-
dation Programme of the UCM, Research Group
920267 and the Iberoamerican CYTED network No.
210RT0398. CM is supported by the Universidad de

Cartagena (Colombia) and the Alban Programme of
High Level Scholarships for Latin America, fellowship
E07D400516CO. We are grateful to Dr Andre
´
s Gar-
cı
´
a-Granados for providing MA and helpful discus-
sions. We thank Susana Pe
´
rez-Benavente for excellent
technical help.
Competing interests
The use of MA as an antiparasitic agent is protected
by a patent owned by the University of Granada (date
of filing, 29 March 2007; Patent Number WO⁄ 2007 ⁄
Maslinic acid targets on Plasmodium falciparum C. Moneriz et al.
2958 FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS
034009). The authors declare no competing financial
interests.
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