Multi-targeted activity of maslinic acid as an antimalarial natural compound ´ Carlos Moneriz1,4, Jordi Mestres2, Jose M Bautista1,3, Amalia Diez1,3 and Antonio Puyet1,3 ´ ´ Departamento de Bioquımica y Biologıa Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain ´ ` Chemogenomics Laboratory, Research Unit on Biomedical Informatics (GRIB), Institut Municipal d’Investigacio Medica and Universitat Pompeu Fabra, Barcelona, Spain ´ Instituto de Investigacion del Hospital 12 de Octubre, Universidad Complutense de Madrid, Spain ´ Departamento de Bioquımica, Facultad de Medicina, Universidad de Cartagena, Colombia Keywords apicomplexa; merozoite surface protein; metalloprotease inhibition; PfSUB1; phospholipase; plasmodium Correspondence ´ A Puyet, Departamento de Bioquımica y ´ Biologıa Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, E28040 Madrid, Spain Fax: +34 913 943 824 Tel: +34 913 943 827 E-mail: apuyet@vet.ucm.es (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 complex, probably by inhibition of PfSUB1 In addition, MA was also found to inhibit metalloproteases of the M16 family by a non-chelating mechanism, suggesting the possible hindrance of plasmodial metalloproteases belonging to that family, such as falcilysin and apicoplast peptide-processing 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 interfere with essential processes at the blood stage of the parasite [1], which accounts for the main clinical symptoms of disease Despite the wide variety of potential targets identified in the intra-erythrocytic cycle of Abbreviations IC50, 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 Maslinic acid targets on Plasmodium falciparum C Moneriz et al P falciparum [2] only a few drugs have found application as therapeutic agents, like those interfering with hemozoin polymerization in the vacuole (chloroquine, quinine, mefloquine and other alkaloids), the dihydrofolate pathway (pyrimethamine, sulphadoxine, proguanil), the mitochondrial electron transport chain (atovaquone) or triggering of oxidative stress (artemisin and derivatives, primaquine) Maslinic acid (MA) is a natural pentacyclic triterpene 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], vasorelaxation [6] and anti-tumoural [7,8] activities, MA has been shown to specifically inhibit glycogen phosphorylase [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, epipomolic and tormentic acids [12] MA appears to display antiparasitic activities in apicomplexa [13], further demonstrated in Toxoplasma gondii cultures, where the likely inhibition of proteolytic activity leads to a reduction 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 intraerythrocytic 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 Plasmodium proteases The main source of amino acids for 2952 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 developed based on the structure of the available inhibitors of cathepsin D [19], a lysosomal protease of mammalian cells, and chalcones and phenothiazines have been assayed as inhibitors of falcipain-2 [20,21] Inhibition of the metalloproteases falcilysin and neutral aminopeptidases acting at the terminal stages of haemoglobin degradation are also considered as potential antimalarial targets [22,23] Other proteases related to the maturation 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 parasites [26] Computational ligand-based approaches to predict the potential affinity of compounds have been developed 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 potential inhibitory activities of MA on P falciparum has been carried out, focusing first on the putative protease inhibition Furthermore a chemogenomic-based screening using MA as ligand to predict its most probable targets was performed, searching for enzymatic activities and protein binding structures which could eventually 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 cysteine, aspartic, serine and metalloproteases The results, shown in Table 1, indicate that MA is a strong inhibitor of the metalloprotease thermolysin, showing also FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS C Moneriz et al Maslinic acid targets on Plasmodium falciparum Table 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 lM Enzyme Protease class Enzyme (mmL)1) Detection limit (mmL)1) IC50 MA (lM) Subtilisin A Thermolysin Papain Pepsin Serine Metallo Cysteine Aspartic 0.4 400 20 5000 0.001 1.5 0.08 0.25 59.3 ± 9.2 ± 3.4 107 ± 12.5 No inhibition low half maximal inhibitory concentration values (IC50) for serine and cysteine proteases Remarkably, no inhibition was observed on the aspartic protease pepsin However, a possible specific inhibition of plasmodial 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 inhibition 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 falciparum belong to the A1 class [32], thus explaining the lack of inhibition observed in the protein extracts Table Effect of MA on aspartic protease activity in P falciparum protein extracts MA and pepstatin A were tested at 300 lM Enzyme or total protein extract Cathepsin D Parasite Schizont Leukocyte Compound % Inhibition None Pepstatin A MA None Pepstatin A MA None Pepstatin A MA 100 100 0 100 15 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 histoaspartic 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 visualization 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 initial 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 globin 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 IC50 in the range of 50 lm Subtilisin is a serine protease of the S8 family closely related to the subtilases (PfSUB1), (PfSUB2) and (PfSUB3) reported in P falciparum [32,40] These proteins play an essential FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS 2953 Maslinic acid targets on Plasmodium falciparum A C Moneriz et al B MW Start rings (0 h) Untreated (24 h) Maslinic acid (24 h) kDa Leupeptin (24 h) 250 148 98 64 50 36 22 16 Fig MA-treated cultures not accumulate undegraded haemoglobin Synchronized ring-stage P falciparum was cultured in the presence of 100 lM MA or 100 lM leupeptin for 18 h, followed by extraction of proteins and parasite visualization in thin blood smears (A) Coomassie 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 haemoglobin 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 role in the erythrocyte invasion by the parasite merozoite through a mechanism involving the discharge of PfSUB1 into the parasitophorous vacuole and the proteolytic 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, corroborating 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 similarities with MA A comparative study on both molecules might help in the design of simpler structures behaving as specific inhibitors of this protease 2954 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 polymorphic variant MSP7-3 has been used to immunize mice against blood stage infection [46] The immunization 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 neutralizing 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 carried 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 FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS C Moneriz et al Maslinic acid targets on Plasmodium falciparum A kDa Control (0 h) Control (12 h) MA (12 h) C H 3C HO O OH OH 53 35 28 CH3 HO HO H CH3 HO H COOH OH O OH O CH3 HO H CH HC 19 B OH OH CH3 207 114 78 OH Maslinic acid Start schizonts (0 h) MTR12113 No drug (12 h) MA (12 h) Fig Inhibition of MSP1 primary processing by MA Highly synchronized early schizonts of P falciparum 3D7 ( 36 h post-invasion) cultured for 12 h in the presence of MA (100 lM) were used to obtain an extract of parasite proteins and Wright’s stained thin smears for monitorization 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 lM 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 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 mitochondrial 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 accumulation 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 inhibition 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 multitargeted 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 compiles the list of six proteins 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 Phospholipase A2 (PLA2) was detected in P falciparuminfected human erythrocytes and found to be inhibited by the antimalarial drugs chloroquine, quinine and FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS 2955 Maslinic acid targets on Plasmodium falciparum C Moneriz et al % zinc chelation 100 Table Inhibition of PLA2 activity by MA MA and 3,4-dichloroisocoumarin (DIC) were tested at 100 and 400 lM The compounds were incubated with the enzyme control and schizonts protein for 15 and h respectively Results are expressed as the percentage of inhibition compared with the control with no inhibitor added 80 60 40 Enzyme or total protein extract 20 20 50 100 200 Concentration (mM) PLA2 bee venom 300 Schizont Fig Zinc chelating assay for MA Percentage of zinc chelation detected using Eriochrome Black T as an indicator of non-complexed zinc cations The assay was carried out by adding different amounts of MA (black bars) or EDTA (grey bars) to a solution containing 32 lM Zn2+ Results are expressed as 100 · A610 nm(sample) ⁄ A610 nm(control without zinc) Table Results of the chemogenomic screening of proteins with high probability of binding to MA Enzyme MA predicted affinity (lM) Phospholipase A2 Protein tyrosine phosphatase CYP2C8 Acetylcholinesterase 5-HT2B Glycogen phosphorylase 3.2 4.0 6.3 10 13 40 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 metabolize 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 receptors in general as potential targets mediating differential 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 2956 Inhibitor Inhibition at 100 lM (%) Inhibition at 400 lM (%) MA DIC MA DIC 16 18 12 13 25 20 15 18 the highest predicted affinity The results obtained are collected in Table 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% inhibition 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 inhibition 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 coverage 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 proteases frequently bind their inhibitors ⁄ substrates in extended or b-strand conformation, requiring a linear arrangement of the substrate backbone [53] The pentacyclic 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 evidence 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 proteases Should these arguments be confirmed experimentally, MA would be a valuable lead molecule for development of specific drugs against apicomplexa parasites FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS C Moneriz et al 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 SigmaAldrich (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) Erythrocytes (RBC) were obtained from type A+ healthy human local donors and collected in tubes with citratephosphate-dextrose anticoagulant (VacuetteÒ Greiner BioOne GmbH, Kremsmunster, Austria) The culture medă 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 lgỈmL)1 gentamicine (Sigma-Aldrich) and 25 mm NaHCO3 (Sigma-Aldrich) Each culture was started by mixing uninfected and infected erythrocytes to achieve a 1% haematocrit and incubated in 5% CO2 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 previously [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 volume of saponin 0.2% in NaCl ⁄ Pi (PBS) 1· and vortexed gently for s to lyse the RBC membranes The released parasites were pelleted at 10 000 g for 10 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 °C for 15 followed by three freeze–thaw cycles After centrifugation (13 000 g, 10 min, °C) the supernatant was collected and referred to as parasite 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 Maslinic acid targets on Plasmodium falciparum Protease inhibition assays The protease activities were conducted in vitro using the internally quenched fluorogenic peptide substrate (EnzChekÒ Protease Assay Kit-E33758; Invitrogen) 50 lL of different protease dilutions, with or without MA at different concentrations, 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 The fluorescence intensity was measured at 485 nm excitation and 528 nm emission using a Perkin Elmer LS-50B luminescence spectrophotometer Background fluorescence was subtracted from the inset data Plots of percentage 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 thermoproteolyticus 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 mm l-cysteine The effect of MA on the activity of aspartic proteases present in parasite protein extracts was assayed spectrofluorometrically with the internally quenched fluorescent substrate MCA-G-K-P-I-L-F-F-R-L-K(DNP)-D-Arg-NH2 trifluoroacetate salt (Sigma-Aldrich), which is not initially fluorescent due to quenching of the 7-methoxycoumarin-4acetyl (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 microplate, 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 samples were incubated at 37 °C for 10 to allow the inhibition of the enzyme After this time, lL of the substrate were added and the samples were incubated at 37 °C for 30 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 LS50B luminescence spectrophotometer Plasmodial MSP processing assay To analyse the possible inhibitory activity of MA on the processing 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 FEBS Journal 278 (2011) 2951–2961 ª 2011 The Authors Journal compilation ª 2011 FEBS 2957 Maslinic acid targets on Plasmodium falciparum C Moneriz et al to allow schizont rupture and merozoite invasion The parasite 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, Waukeha, WI, USA) at a : 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 accumulation 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 cultures, 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 inhibitors as indicated were collected after 18 h incubation, and proteins from parasite extracts were obtained as described above, solubilized in electrophoresis sample buffer and electrophoresed through 15% SDS ⁄ PAGE [36,37] Proteins were identified by staining the gel with Coomassie Blue 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 lL of parasite protein extract or 10 lL of PLA2 bee venom supplied by the kit as positive control, and lL of the corresponding 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(heptanoylthio)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 CaCl2, 100 mm KCl and 0.3 mm Triton X-100 Chelating activity assay The metal chelating activity was measured using the complexometric indicator Eriochrome Black T (Sigma-Aldrich) Samples were prepared by mixing 100 lL of 100 lm ZnSO4.H2O, 200 lL of 0.3 m Na2CO3 pH 10, 10 lL of compound (MA or EDTA as control) and lL of Eriochrome Black T (5 mgỈmL)1 in ethanol, pH 10) The pH of the Eriochrome Black T solution was adjusted by adding buffer solution dropwise until the colour changed from purple to blue 200 lL of each sample were transferred to a 96-well microplate 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 activity compared with a control without zinc EDTA was used as a positive control for chelating activity In silico target screening Our in silico target screening approach relies on the assumption that the set of bioactive ligands collected for a given target provides a complementary description of the target from a ligand perspective In order to be able to process this information 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 chemical structures with a different degree of fuzziness and thus complementing each other in terms of structural congenericity 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] Acknowledgements This work was supported by grants from the Spanish Ministry of Education and Science (BIO2007-67885 and BIO2010-17039) and the Research Teams Consolidation 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 Andres Garcı´ a-Granados for providing MA and helpful discus´ sions We thank Susana Perez-Benavente for excellent technical help Competing interests 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 2958 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 ⁄ FEBS Journal 278 (2011) 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investigated as possible targets for specific antimalarial. .. (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... 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 histoaspartic protease), cysteine proteinases