Activity against Plasmodium falciparum of cycloperoxide compounds obtained from the sponge Plakortis simplex

Ernesto Fattorusso1, Silvia Parapini2, Claudio Campagnuolo1, Nicoletta Basilico2, Orazio Taglialatela-Scafati1 and Donatella Taramelli2,*

1 Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli ‘Federico II’, via D. Montesano 49, I-80131 Napoli; 2 Istituto di Microbiologia, Università degli Studi di Milano, Via Pascal 36, 20133 Milano, Italy

Received 8 February 2002; returned 24 June 2002; revised 31 July 2002; accepted 22 September 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is an urgent need to discover new antimalarials, due to the spread of chloroquine resistance and the limited number of available drugs. In the last few years, artemisinin, the endoperoxide sesquiterpene lactone derived from Artemisia annua, and its derivatives proved to be very active against Plasmodium falciparum. These compounds are characterized by an endoperoxide pharmacophore that is critical for their antimalarial activity. There are several reports, from our group and others, that marine organisms can be another natural source of stable cyclic peroxides, with selective antifungal or antibacterial activity. With the aim of identifying new bioactive molecules, we evaluated in vitro the antimalarial activity of the major cycloperoxides extracted from the sponge Plakortis simplex. The six-membered endoperoxide compounds plakortin and dihydroplakortin, but not the five-membered cycloperoxide plakortide E, inhibited the growth of cultured P. falciparum parasites, both chloroquine-sensitive D10 strain and chloroquine-resistant W2 strain. The IC50 values were similar for both compounds and in the range of 1263–1117 nM against D10, and 735–760 nM against W2, using the colorimetric parasite lactate dehydrogenase assay. The activity of plakortin and dihydroplakortin was significantly higher against chloroquine-resistant than chloroquine-susceptible parasites, following a pattern similar to that of artemisinin, although they were 50-fold less active. Moreover, plakortin and dihydroplakortin showed an additive effect when used in combination with chloroquine. These results support further studies on cycloperoxides of marine origin to characterize their mechanism of action and identify/synthesize new compounds with stronger antimalarial activity.

Keywords: P. falciparum, artemisinin, chloroquine, peroxides, marine metabolites, antimalarials


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Malaria continues to be a major cause of morbidity and mortality in tropical countries. The reality is probably worse than traditionally cited figures: a recent analysis estimates, at a minimum, between 700 000 and 2.7 million deaths each year from malaria (>75% of them African children) and between 400 and 900 million acute febrile episodes per year in African children under the age of five living in malaria-endemic regions.1 Part of the reason for the failure to control malaria is the emergence and spread of resistance to first-line antimalarial drugs, cross-resistance between the members of the limited number of drug families available, and in some areas, multidrug resistance.2

A major breakthrough of the past decades has been the discovery by Chinese researchers of artemisinin (qinghaosu), an endoperoxide sesquiterpene lactone, as the active component of Artemisia annua, a herbal remedy used in Chinese folk medicine for 2000 years. This molecule and its oil-soluble (e.g. artemether and arteether) and water-soluble (e.g. artesunate) semi-synthetic derivatives have shown excellent anti-Plasmodium efficacy in vitro and are being used increasingly, especially in combination with traditional antimalarials (e.g. mefloquine).3

These compounds are structurally distinct from other antimalarial drugs in that they are characterized by a peroxide bridge embedded in a 1,2,4-trioxane (six-membered ring containing three oxygen atoms) pharmacophore, which is deemed essential for their antimalarial activity, since the corresponding acyclic compounds lacking the peroxidic bridge are biologically inactive.4

The research in this field continues for novel peroxide-bridged compounds, particularly with a view to obtaining structurally more simple and thus synthetically more accessible analogues (e.g. simple trioxanes5 or tetraoxanes6), or to render them more stable under physiological conditions (e.g. artelinic acid) or safer (artemisinin and derivatives, as well as arteether, have shown some neurotoxic effects).7 One of the strategies is to find other natural sources of stable peroxides.

Marine sponges continue to attract attention as a rich source of structurally novel bioactive secondary metabolites. In particular, a number of cyclic peroxides have been isolated from organic extracts of marine sponges belonging to the genera Sigmosceptrella, Latrunculia, Mycale, Diacarnus, Chondrilla, Xestospongia, Plakinastrella and Plakortis. Sponges of the first four genera produce mainly norsesterterpene and norditerpene peroxide acids and some of them, such as sigmosceptrellin-A, and its C-3 epimer sigmosceptrellin-B, have been shown to possess in vitro activity against Plasmodium.8 On the other hand, cyclic peroxides contained in sponges of the Plakinidae family (Plakinastrella and Plakortis), which are prominent members of both Caribbean and Indo-Pacific coral reefs, have been identified as polyketide metabolites containing six- or five-membered 1,2-dioxygenated rings. In particular, starting from the isolation of the parent compounds plakortin9 and chondrillin,10 Plakortis sponges have been recognized as prolific sources of 1,2-dioxane, 1,2-dioxolane and 3-alkoxy-1,2-dioxine (peroxyketal) compounds. The antimalarial activity of these classes of metabolites has not yet been fully assessed.

We recently examined the apolar extracts of the Caribbean sponge Plakortis simplex and isolated plakortin (1) (in remarkable amounts), along with other novel (e.g. dihydroplakortin, 2) and known (e.g. plakortide E, 3)11 cyclic peroxides. In addition, we recently elucidated the absolute configuration at the four chiral carbons of plakortin and dihydroplakortin,12 as reported in Figure 1. For plakortide E only the relative stereochemistry around the dioxolane ring is known (Figure 1). Most cycloperoxide acids have shown antifungal and/or antibacterial activity, but limited cytotoxic activity against mammalian cells.12 As an extension of such work, we are herein reporting on the antimalarial activity of the major endoperoxide metabolites plakortin, dihydroplakortin and plakortide E.



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Figure 1. Chemical structures of the cycloperoxides under study and related compounds. Plakortin (1), dihydroplakortin (2), plakortide E (3), peroxyplakoric acid B3 (4), plakortide I (5) and plakortide L (6).

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents

RPMI 1640 medium was purchased from Gibco-BRL, and human A-positive red blood cells and plasma were kindly provided by the Blood Bank of the National Cancer Institute, Milano, Italy. Chloroquine diphosphate (CQ) (C-6628), artemisinin (36,159-3), ethanol and DMSO were all obtained from Sigma-Aldrich, Milan, Italy.

Test compounds

A specimen of P. simplex (57 g, dry weight after extraction) collected in 1998 along the coasts of Berry Island (Bahamas) was homogenized and extracted with methanol (4 x 500 mL) and chloroform (4 x 500 mL). The methanol extract was partitioned between H2O and n-BuOH and then the organic phases were combined and concentrated in vacuo. The organic phase (29.3 g) was subjected to chromatography on a column packed with RP18 silica gel and eluted with a system of solvents of decreasing polarity from H2O to MeOH/H2O 9:1. Fractions eluted with MeOH/H2O 8:2 and 9:1 were combined (12.0 g) and further chromatographed by medium-pressure liquid chromatography (MPLC) over silica gel (230–400 mesh; solvent gradient system of increasing polarity from n-hexane to MeOH). Fractions eluted with n-hexane/EtOAc 9:1 and 8:2 were rechromatographed separately by high-performance liquid chromatography (HPLC) (eluent n-hexane/EtOAc 95:5) leading to the recovery of plakortin (1, 2.25 g), dihydroplakortin (2, 260 mg) and plakortide E (3, 100 mg) in a pure state (Figure 1). MPLC was performed using a Büchi 861 apparatus with RP18 and SiO2 stationary phases. HPLC separations were achieved on a Beckman apparatus equipped with RI detector and LUNA SI60 (250 x 4 mm) columns. All the solvents used were provided by Sigma-Aldrich.

Parasite cultures

P. falciparum cultures were carried out according to the method described by Trager and Jensen.13 Briefly, the CQ-susceptible (CQ-S), moderately mefloquine-resistant clone D10 and the CQ-resistant (CQ-R), mefloquine-susceptible clone W2 were maintained at 5% haematocrit (human type A-positive red blood cells) in complete culture medium at 37°C. Complete medium contained RPMI 1640 medium (Gibco-BRL; 24 mM NaHCO3) with the addition of 10% heat-inactivated A-positive human plasma, 20 mM HEPES (Biological Industries, Kibbutz, Israel), 2 mM glutamine (Biological Industries). All the cultures were maintained in a standard gas mixture consisting of 1% O2, 5% CO2, 94% N2. When parasitaemia exceeded 5%, subcultures were taken; the culture medium was changed every second day.

Parasite growth and drug susceptibility assay

Compounds were dissolved in either water (chloroquine), ethanol (artemisinin) or DMSO (compounds 13) and then diluted with medium to achieve the required concentrations (in all cases the final concentration contained <1% ethanol or DMSO, which were found to be non-toxic to the parasite). Drugs were placed in 96-well flat-bottomed microplates (Costar 3596) and 10 two-fold dilutions were made starting at 1 µg/mL for all the compounds tested, except for artemisinin, the initial concentration of which was 100 ng/mL. Asynchronous cultures with parasitaemia of 1–1.5% and 1% final haematocrit were aliquotted into the plates and incubated for 72 h at 37°C. Parasite growth was determined spectrophotometrically by measuring the activity of the parasite lactate dehydrogenase (pLDH), in control and drug-treated cultures according to the method originally described by Makler & Hinrichs.14 The pLDH activity is distinguishable from host LDH using the 3-acetyl pyridine adenine dinucleotide (APAD) as co-factor. Briefly, at the end of the incubation, the cultures are carefully resuspended, and aliquots of 20 µL are removed and added to 0.1 mL of the Malstat reagent in a 96-well microtitre plate. The Malstat reagent is made with 0.125% Triton X-100, 130 mM L-lactic acid, 30 mM Tris buffer and 0.62 µM APAD. The spectrophotometric assessment of pLDH activity is facilitated by adding 25 µL of a solution of 1.9 µM NBT (Nitro Blue Tetrazolium) and 0.24 µM PES (phenazine ethosulphate) to the Malstat reagent. As APADH is formed, the NBT is reduced and forms a blue formazan product that can be measured at 650 nm. The antimalarial activity of the test compound was expressed as the IC50; each IC50 value is the mean ± S.D. of at least three separate experiments performed in triplicate. Statistical analysis was performed using the paired t-test with Statview software.

The effect of the cycloperoxides on the IC50 of chloroquine was determined by potentiation tests as described previously.15 Isobolograms were constructed by plotting a pair of fractional IC50s for each combination of chloroquine and cycloperoxides. Chloroquine fractional IC50 was calculated by dividing the IC50 of chloroquine combined with each cycloperoxide by the IC50 of chloroquine alone, and these data were plotted on the horizontal axis. The corresponding cycloperoxide fractional IC50s were calculated by dividing each fixed concentration by the IC50 of the cycloperoxides alone, and was plotted on the vertical axis. An isobologram close to the diagonal indicates an additive effect. Curves significantly above or below the diagonal indicate antagonistic or synergic effects, respectively.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Using the pLDH assay, plakortin (1), dihydroplakortin (2) and plakortide E (3) were assayed against D10, CQ-S strain and W2, CQ-R strain of P. falciparum. A representative dose–response curve is reported in Figure 2; compounds 1 and 2 showed almost identical antimalarial activity against both P. falciparum strains, whereas compound 3 was inactive. When compared with CQ, the activity of plakortin (1) and dihydroplakortin (2) was 1.6- and 42-fold lower than CQ in the W2 and D10 strains, respectively.



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Figure 2. Dose–response curve of the effect of plakortin (1), dihydroplakortin (2), plakortide E (3) and chloroquine on the growth of D10, CQ-S (a) and W2, CQ-R (b) strains of P. falciparum. Each point represents the mean ± S.D. from one representative experiment in triplicate.

 
The IC50 values obtained for compounds 1, 2, chloroquine and artemisinin for P. falciparum D10 and W2 strains are reported in Table 1. Compounds 1 and 2 were more active against the CQ-R than the CQ-S strain. When the results were averaged from eight different experiments, the IC50 of plakortin (1) and dihydroplakortin (2) for the CQ-R strain was significantly different from the IC50 for the CQ-S strain (P < 0.05). The response pattern is similar to that of artemisinin, although the IC50s differ by ~50-fold.


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Table 1.  IC50 values of different compounds tested against CQ-S, D10 and CQ-R, W2 strains of P. falciparum
 
We then looked for a possible potentiation of CQ activity by adding different doses of plakortin (1) and dihydroplakortin (2). Both resulting isobolograms (Figure 3a and b) are close to the diagonal, therefore indicating that only an additive effect was obtained, with the activity of CQ mainly unchanged in the presence of plakortin (1) or dihydroplakortin (2). The slight antagonistic effect observed at very low doses between plakortin (1) and CQ against the D10, CQ-S strain did not reach statistical significance (Figure 3a).



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Figure 3. Isobolograms of in vitro drug interaction between plakortin (a) and dihydroplakortin (b) with chloroquine against CQ-S, D10 and CQ-R, W2 strains of P. falciparum. Each point in the isobolograms was obtained by dividing the IC50 of chloroquine plus test compounds by the IC50 of chloroquine alone (abscissa) and by dividing the fixed drug concentration by the IC50 of its intrinsic activity (ordinate). Results are the mean of five independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this work we evaluated the in vitro antimalarial activity of the major cycloperoxides extracted from the sponge P. simplex. The two simple six-membered endoperoxide compounds plakortin (1) and dihydroplakortin (2) exhibit bioactivity in the nM range, whereas, interestingly, the structurally related five-membered cycloperoxide plakortide E (3) is practically inactive. In addition, the presence of the double bond in one of the side chains of plakortin is irrelevant for the bioactivity, as indicated by the identical potency of plakortin and its 9,10-dihydro derivative.

Plakortin (1) and dihydroplakortin (2) exhibited antimalarial activity against both CQ-R and CQ-S P. falciparum clones. The IC50 was significantly lower (efficacy was higher) for the CQ-R than the CQ-S strain (P < 0.05 by the paired t-test). A similar pattern of reactivity is reported for artemisinin, although compounds 1 and 2 are almost 50-fold less active. These findings suggest that these compounds, like other antimalarial cycloperoxides, do not share the mechanism of resistance of CQ and could be investigated further to treat CQ-R parasites.

Moreover, when tested in combination, we found no evidence of synergy between chloroquine and the two cycloperoxides. Under the conditions of the assay, additive effects against both CQ-S and CQ-R strains of P. falciparum were obtained. A slight, although not significant, antagonism between low doses of plakortin and CQ was seen only against the D10 strain. This is in line with various reports describing the interactions between conventional drugs and natural extracts. Whereas a combination of artemisinin or artemether with CQ proved to be additive or antagonistic, plant-derived compounds have been reported to either increase or antagonize the activity of CQ, or simply showed additive effects, such as our samples.16,17 How the results of such in vitro findings extrapolate to the treatment of clinical malaria is unclear.

The mechanism of action of plakortin as well as of other simple endoperoxides is not known. Concerning artemisinin, there is strong evidence to suggest that its endoperoxide group reacts with a haem iron centre initially, giving rise to an oxyl radical that then rearranges to produce C-centred radicals. These latter species would be the final entities responsible for anti-Plasmodium activity, functioning as alkylating agents towards the macromolecules within the protozoan.18 The presence of a non-peroxidic oxygen atom is thought to play a substantial role in facilitating these steps; in fact the carba-artemisinin analogue, which possesses a simple peroxide pharmacophore with the non-peroxidic oxygen atom in the trioxane pharmacophore replaced by a carbon atom, displays activity against P. falciparum 25-fold less than that of artemisinin.19 It should be noted that the above mechanism of action is not yet fully agreed upon, as there is no unequivocal evidence as to whether reductive scission or peroxide ring opening occurs initially. The difference in chemical structure (dioxane versus trioxane) could partly explain the different antimalarial activity of plakortin (1) and dihydroplakortin (2) compared with artemisinin.

Very recently, some papers reporting on the antimalarial activity of simple monocyclic 1,2-dioxane derivatives have appeared. Synthetic and natural 3-alkoxy-1,2-dioxine and 3-alkoxy-1,2-dioxane (peroxyketal) derivatives (e.g. 4) were shown to possess good antimalarial activity.20 In both these classes of molecules the substituent at position 3 could partly mimic the non-peroxidic oxygen atom of artemisinin. Good antimalarial activity was also recently reported for a 1,2-dioxane derivative substituted at position 3 with an {alpha},ß unsaturated ketone (5), and, interestingly, the corresponding molecule lacking the carbonyl function (6) was completely inactive.21 The above data on the monocyclic 1,2-dioxane derivatives seemed to be indicative of a key role of the functionalization at C-3, in addition to the endoperoxide bond. Apparently, this evidence is in contrast to our present data indicating significant antimalarial activity of plakortin (1) and dihydroplakortin (2), both lacking functionality at C-3.

Our efforts are currently focused both on the isolation of novel lead compounds from natural sources and on the synthesis of bioactive natural molecules (plakortin and dihydroplakortin) and of their analogues in order to investigate the mechanism of action of these simple endoperoxide derivatives.


    Acknowledgements
 
We wish to thank Dr Piero Olliaro from WHO/TDR, Geneva, Switzerland, for helpful suggestions and critical reading of the manuscript. We also wish to thank Professor Joseph R. Pawlik for giving us the opportunity to participate in an expedition to the Caribbean Sea, during which the sponge P. simplex was collected, and Professor M. Pansini (Istituto di Zoologia, Università di Genova, Italy) for identifying the organism. This work was supported by Ministero Italiano dell’Università e della Ricerca Scientifica e Tecnologica, Co-finanziamento 2001/061849 to D.T. and by ‘M.U.R.S.T., PRIN Chimica dei Composti Organici di Interesse Biologico’ to E.F.


    Footnotes
 
* Corresponding author. Tel: +39-02-5031-5071; Fax: +39-02-5031-5068; E-mail: donatella.taramelli{at}unimi.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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