Anti- Plasmodium activity of ceramide analogs
© Labaied et al. 2004
Received: 30 October 2004
Accepted: 10 December 2004
Published: 10 December 2004
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© Labaied et al. 2004
Received: 30 October 2004
Accepted: 10 December 2004
Published: 10 December 2004
Sphingolipids are key molecules regulating many essential functions in eukaryotic cells and ceramide plays a central role in sphingolipid metabolism. A sphingolipid metabolism occurs in the intraerythrocytic stages of Plasmodium falciparum and is associated with essential biological processes. It constitutes an attractive and potential target for the development of new antimalarial drugs.
The anti- Plasmodium activity of a series of ceramide analogs containing different linkages (amide, methylene or thiourea linkages) between the fatty acid part of ceramide and the sphingoid core was investigated in culture and compared to the sphingolipid analog PPMP (d,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). This analog is known to inhibit the parasite sphingomyelin synthase activity and block parasite development by preventing the formation of the tubovesicular network that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite.
Analogs containing methylene linkage showed a considerably higher anti- Plasmodium activity (IC50 in the low nanomolar range) than PPMP and their counterparts with a natural amide linkage (IC50 in the micromolar range). The methylene analogs blocked irreversibly P. falciparum development leading to parasite eradication in contrast to PPMP whose effect is cytostatic. A high sensitivity of action towards the parasite was observed when compared to their effect on the human MRC-5 cell growth. The toxicity towards parasites did not correlate with the inhibition by methylene analogs of the parasite sphingomyelin synthase activity and the tubovesicular network formation, indicating that this enzyme is not their primary target.
It has been shown that ceramide analogs were potent inhibitors of P. falciparum growth in culture. Interestingly, the nature of the linkage between the fatty acid part and the sphingoid core considerably influences the antiplasmodial activity and the selectivity of analogs when compared to their cytotoxicity on mammalian cells. By comparison with their inhibitory effect on cancer cell growth, the ceramide analogs might inhibit P. falciparum growth through modulation of the endogenous ceramide level.
Until recently, sphingolipids were primarily considered to be structural components of membranes. However, data accumulated during the last decade have expanded the view of their biological functions. They are now also considered to be key molecules which regulate many functions essential to eukaryotic cells [1–5]. They are involved, for example, in the regulation of membrane fluidity and are part of discrete membrane microdomains or rafts implicated in signalling and trafficking in cells [4, 6–8]. Interest in sphingolipids was strengthened by an increasing body of evidence demonstrating their role as secondary messengers for intracellular signal transduction pathways that regulate many cellular processes. For example, ceramide accumulates in response to several different inducers such as cytokines, cytotoxic agents or to stressful conditions, which lead to cell cycle arrest or to apoptosis . Sphingosine is a protein kinase C inhibitor  that inhibits growth or stimulates proliferation, depending upon the cell type [11, 12].
Ceramide plays a central role in sphingolipid metabolism . It can be converted into SPM through transfer of the choline phosphate group from phosphatidylcholine or serves as a precursor for complex sphingolipids (cerebrosides which possess sugar residues and gangliosides which contain sialic acid residues in addition to the carbohydrate units).
Moreover, ceramide can be phosphorylated by a distinct kinase and can also be produced by enzymatic hydrolysis of complex sphingolipids. In turn, ceramide can be hydrolyzed to sphingosine and fatty acid by ceramidases.
In contrast to yeast and mammalian cells, the current understanding of sphingolipid metabolism and the biological role of sphingolipids in the development of Plasmodium falciparum, the causative agent of malaria, is still limited. Gerold et al.  provided evidence that de-novo synthesis of sphingolipids occurs in the intraerythrocytic stages of the human malaria parasite P. falciparum and can be inhibited by the well established inhibitors of de-novo ceramide biosynthesis, fumonisin B1, cyclo-serine and myriocin [15, 16]. However, these compounds are weak inhibitors of parasite growth. Evidence was provided that another pathway for the synthesis of glycosylated sphingolipids exists in P. falciparum [14, 17]. The importance of sphingolipid metabolism for parasite development was demonstrated by Haldar's work showing that: (i) The parasite contains two distinct forms of SPM synthase, one sensitive to sphingolipid analogs, d,1-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) or d,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (Figure 1), known to inhibit the synthesis of glucosylceramide in mammalian cells , and the second insensitive to them ; (ii) These analogs blocked the parasite proliferation in culture by preventing the formation of the tubovesicular network (TVN) that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite [20–22]. Neutral magnesium-dependent sphingomyelinase activity was also identified in P. falciparum [23–25], indicating that a sphingomyelin cycle (ceramide-SPM conversion) exists in Plasmodium. Recently, an increase in the intracellular ceramide content and an activation of parasite sphingomyelinase(s) were found to be associated with the parasite death process as induced by artemisinine and mefloquine .
Given the importance of sphingolipids in many cellular functions and the central role of ceramide in sphingolipid metabolism, the anti- Plasmodium activity of non-natural analogs of ceramides was investigated on the intraerythrocytic development of P. falciparum. Interestingly, a series of analogs containing a methylene (CH2-NH) linkage between the fatty acid and the sphingoid-analog core showed considerably higher anti- Plasmodium activity than their counterparts with a natural amide (CO-NH) linkage or than PPMP. The methylene analogs irreversibly blocked parasite development in contrast to PPMP whose effect is cytostatic. Their efficiency in inhibiting parasite growth did not correlate with their potential to inhibit parasite SPM synthase activity, indicating that SPM synthase is not their primary target. Possible mechanisms of action are discussed.
P. falciparum strains (FcB1/Colombia, K1/Thailand, F32/Tanzania, W2/Indochina) were maintained in continuous culture on human erythrocytes in RPMI medium containing 7% (v/v) heat-inactivated human serum under an atmosphere of 3% CO2, 6% O2, 91% N2, at 37°C, as described by Trager and Jensen . Parasite synchronization was performed successively by treatment with 5% (w/v) sorbitol and by concentration in gelatin solution as previously described .
Drug susceptibility assays were performed using a modification of the semi automated microdilution technique of Desjardins et al. . Stock solutions of test compounds were prepared in DMSO. Drug solutions were serially diluted twofold with 100 μl culture medium in 96-well plates. Asynchronous parasite cultures (100 μl, 1 % parasitemia and 1 % final hematocrite) were added to each well and incubated for 24 hours at 37°C prior to the addition of 0.5 μCi of [3H] hypoxanthine (Amersham, France, 1 to 5 Ci.mmol/ml) per well. After a further incubation of 24 hour, plates were frozen and thawed. Cell lysates were then collected onto glass-filter papers and counted in a liquid scintillation spectrometer. The growth inhibition for each drug concentration was determined by comparison of the radioactivity incorporated in the treated culture with that in the control culture (having the same final % of DMSO) maintained on the same plate. The concentration causing 50% growth inhibition (IC50) and 90% growth (IC90) were obtained from the drug concentration-response curve and the results were expressed as the means ± the standard deviations determined from several independent experiments. The DMSO concentration never exceeded 0.1% (v/v) and did not inhibit the parasite growth.
A human diploid embryonic lung cell line (MRC-5, Bio-Whittaker 72211D) was used to assess the cytotoxic effects towards eukaryotic host cells. MRC-5 cells were seeded at 5,000 cells per well in 100 μl. After 24 hours, the cells were washed and two-fold dilutions of the drug were added in 200 μl standard culture medium (RPMI medium + 5% fetal calf serum) and maintained for five days under 5% CO2 atmosphere. The final DMSO concentration in the culture remained below 0.1%. Untreated cultures were included as controls. The cytotoxicity was determined using the colorimetric MTT assay according to the manufacturer's recommendations (Cell proliferation kit I, Roche Applied Science, France) and scored as a percentage of reduction in absorption at 540 nm of treated cultures versus untreated control cultures. IC50 values were obtained from the drug concentration-response curve. The results were expressed as the mean ± the standard deviations determined from several independent experiments. The index of selectivity was defined as the ratio of the IC50 value on MRC-5 to that of P. falciparum.
Synchronized cultures (1–2% parasitemia) at the ring stage (0–10 hours old parasites), the trophozoite stage (25–35 hours old parasites) and the schizonte stage (40–48 hours old parasites) were maintained in the presence of drug concentrations in the vicinity of IC50 values. Aliquots were removed at the indicated times, washed three times with culture medium and maintained in culture in the absence or in the presence of a given drug. Parasite morphology was determined on Giemsa-stained smears defined according to the following criteria: the ring stage, when parasites exhibited a peripheral cytoplasm stained by Giemsa and a unstained intraparasitic vacuole; the trophozoite stage, when parasites showed a fully stained cytoplasm, haemozoin crystals and one nucleus; the schizont stage, when parasites presented several distinctive nuclei. Parasitaemias were determined by counting 3,000 cells for each sample. Controls consisted of parasites incubated with DMSO instead of drugs processed in the same way.
SPM synthase activity was measured as described by Haldar et al. . Briefly, assays were performed on P. falciparum cultures at the trophozoite stage (20–30 h old parasites). 400 μl of culture (1 × 108 parasites) were incubated for 60 min at 37°C with 10 μM NBD-C6-ceramide and 0 to 500 μM PPMP or AD2646. Cells were then lysed by freezing and thawing of the culture. Lipids were extracted by a modification of the method of Bligh and Dyer . To each sample, three volumes of a CH3OH/CHCl3 mixture (1:2, v:v) were added and the mixture vortexed for one min. Organic and aqueous phases were separated by centrifugation (12,000 × g, five min) and the organic phase was dried. Lipids were dissolved in 15 μl ethanol and analysed by thin layer chromatography on HPTLC plates (Silica gel 60 F254, Merck, Darmstadt, Germany) in CH3OH/CH3Cl3/NH4OH (75:25:4, v:v:v). For qualitative analyses, the fluorescent lipids were detected under UV and for quantitative analyses, the fluorescent lipid spots were scraped, eluted in one ml methanol and quantified at an excitation of 470 nm and an emission of 530 nm in a spectrofluorometer. The percentage of SPM synthase activity for each drug concentration was determined by comparison of the fluorescence quantified in the analog-treated culture with that in the control culture (without drug).
Infected erythrocytes treated with or without ceramide analogs were incubated for 30 min, at 37°C, in culture medium containing 10 μM BODIPY-FL-C5-ceramide, washed three times with culture medium without serum and fixed overnight, at 4°C, in 3.7% formaldehyde/0.05% glutaraldehyde. Cells were mounted on poly-L-lysine coated slides and viewed using a Nikon Eclipse TE 300 DV inverted microscope with an 100X oil objective mounted on a piezzo electric device using appropriate fluorescence emission filters. Image acquisition (z-series) was performed with a back illuminated cooled detector (CCD EEV: NTE/CCD-1024-EB, Roper Scientific, France) using a 0.2 μm step. Data acquisition and image deconvolution process were performed with Metamorph software (Universal Imaging Corporation, Roper Scientific, France). The images presented correspond to the maximum intensity projection of the deconvoluted z-series.
Non-natural analogs of ceramides were synthesized comprising two functional groups  : 1) A phenyl group substituted on carbon 3 of a sphingoid-like backbone; with the phenyl group replacing the sphingosine acyl chain [33, 34] to which were linked nitro or amine groups, or carbon chains of varying lengths; and 2) a fatty acid with an amide (CO-NH) linkage (series A, Figure 2), a methylene (CH2-NH) or a thiourea (CS-NH) linkages (series B, Figure 3) on carbon 2. Analogs in which the alkyl group replaces the amide were investigated because the carbonyl group of ceramide was shown not to be necessary for triggering apoptosis in mammalian cells. In fact, replacement of the carbonyl group of ceramide by a methylene group substantially reduced the time required for cell death . Only D/L - threo enantiomers were investigated on P. falciparum since reports demonstrated that D/L-erythro enantiomers of ceramide analogs were less efficient in inhibiting glucosylceramide synthase in mammalian cells  and did not inhibit SPM synthase activity in P. falciparum .
Figure 2 and Figure 3 show the IC50 values obtained for the different compounds on the development of the chloroquine-resistant strain FcB1 of P. falciparum in culture (IC50 value for chloroquine = 115 ± 25 nM, n = 3). Interestingly, the nature of the linkage considerably influences the anti- Plasmodium activity. Analogs with amide linkage were found to inhibit parasite growth with IC50 values in the micromolar range (Figure 2). Best IC50 values were similar to that obtained with the ceramide-related compound PPMP (IC50 = 9.0 ± 1.7 μM, n = 3). However, this IC50 value for PPMP differed from the previously reported value (IC50 = 0.85 μM) . The discrepancy may be due to drug susceptibility assay conditions which were performed on synchronized cultures at the ring stage for Lauer et al.  and on asynchronous cultures in the present study. Analogs with methylene linkages were more efficient than the amide analogs in killing parasites with IC50 values in the nanomolar range (Figure 3).
For the D-threo nitro phenyl analogs of series A, no particular increase of the inhibitory activity was observed with the increase of the N -acyl chain length (IC50 values ranging from 10.8 to 40.4 μM, Figure 2). For the series B, best activities were observed for N -alkyl chain length of 12–16 carbons (IC50 values ranging from 17 to 42 nM for the series B, Figure 3). In both series, substitution of the nitrophenyl group by an aminophenyl group instead of nitro group decreased the anti- Plasmodium activity significantly (compare compounds AD2495 and AD2623 of series A, Figure 2; and compounds AD2646 and AD2672 of series B, Figure 3).
Increase of the analog hydrophobicity by substitution of the nitro group of the phenyl ring by alkyl chains seems to decrease the anti- Plasmodium activity of compounds of both series (compare compounds AD2583 and AD2603-7, Figure 2 and compounds AD2646 and AD2677-78-80, Figure 3). Surprisingly, in the B series, the anti- Plasmodium activity was restored in compounds with symmetrical alkyl chains of 6–8 carbon length (compounds AD2651 and AD2670, Figure 3). No systematic difference in anti- Plasmodium activity was observed between D-threo and L-threo enantiomer of a same analogue: e.g. the enantiomers AD2646 and AD2645 of the B series showed similar activity (Figure 3). It can also be noted that ceramide analogs containing a thiourea linkage also showed a significant anti- Plasmodium activity (Figure 3, compounds AD2215-17) with, however, a less pronounced inhibitory effect than analogs with a methylene linkage.
Inhibition of parasite growth by the methylene analog AD2646 was observed having similar IC50 values on the P. falciparum strains K1 (IC50 = 45 nM), F32 (IC50 = 21 nM) and W2 (IC50 = 28 nM), suggesting that the drug is not restricted to a specific strain and acts through a conserved mechanism in malarial parasites. Furthermore, analysis of drug combination with antimalarial drugs showed that AD2646 has a non-synergistic and non-antagonistic effect with CQ on the CQ-resistant strain K1, and with mefloquine and with artemether on the FcB1 strain (data not shown). Compound AD2646 (Figure 1) was selected to further investigate the biological effects of methylene analogs on parasite development.
Structure-activity relationship around AD2646 showed that the presence of a nitro group linked to the phenyl is not essential for anti- Plasmodium activity (Figure 4, compare IC50 values of compounds AD2646 and AD2730) nor hydroxylation on carbon 1 (compare compounds AD2730 and AD2724). In contrast, hydroxylation of carbon 3 is important for anti- Plasmodium activity since removal of the hydroxyl group reduced the activity 13.5 times (compare compounds AD2730 and AD2729).
Cytotoxicity of methylene analogs and PPMP on human MRC-5 cells
Index of selectivity
It must be emphasized that the amide linkage of ceramide analogs is not required for activating apoptosis in cancer cells . An increase of cytotoxicity of ceramide analogs in methylene linkage compared to their counterparts in amide linkage was also observed on the human histolytic lymphoma U937  and the human leukaemia HL-60 cells  however, with higher IC50 values than that observed with P. falciparum.
Development of the ring stage was slightly affected by a continuous incubation with 30 nM AD2646. In contrast, when incubated with 100 and 250 nM, parasite growth was irreversibly blocked at the young trophozoite stage and the parasite degenerated. Drug removal after 24 hours of incubation did not allow a recovery of parasite growth (Figure 5A). The trophozoite stages were more sensitive to AD2646 since a continuous incubation with 30 nM completely blocked development. Parasites did not enter into division and then degenerated. Only a partial recovery of parasite growth was observed when drug was removed after 11 hours of incubation. A more marked effect was observed with 100 nM AD2646 with degenerated parasites already observed after only 11 hours. No recovery of parasite growth was then observed after drug removal (Figure 5B). The schizont stage appeared less sensitive than the trophozoite stage since a slight effect was only observed on the parasite development with 30 nM AD2646. However, parasite growth was irreversibly blocked by an incubation with 100 nM AD2646 and parasites degenerated (Figure 5C). Similar results were observed for the methylene analogs AD2651 and AD2670, the trophozoite stage being the most sensitive with a complete inhibition of parasite development for 250 nM (data not shown).
It can be noted that, in contrast to methylene analogs, addition of PPMP to parasite culture led to a preferential and reversible arrest of parasite development at the ring stage. The schizont stage (>30 hours old parasites) was insensitive to this concentration of drugs [14, 19]. A cytostatic effect of PPMP on the ring-stage was effectively observed : rings blocked by a 24 hours incubation with 5 μM PPMP recovered to a normal growth after drug removal (data not shown). Blockage of parasite development was associated with the inhibition of a sensitive SPM synthase and TVN formation that delivers extracellular nutrients to the parasite [20–22].
These data do not support the hypothesis of parasite growth inhibition due to an inhibition of the parasite SPM synthase activity as was demonstrated for PPMP [19–22] : 1) The anti- Plasmodium activity of AD2646 does not correlate with its inhibitory activity on the SPM synthase. Although AD2646 and PPMP showed similar inhibitory activity on this enzymic activity in parasites in cultures, AD2646 is about 300 times more efficient in inhibiting parasite development than PPMP; 2) In contrast to PPMP which inhibits the parasite development preferentially and reversibly at the ring stage , AD2646 inhibited parasite development preferentially and irreversibly at the trophozoite stage (Figure 5); 3) Inhibition of the SPM synthase activity by PPMP is associated with an inhibition of the TVN formation [19–22]. This was not observed in the presence of AD2646 (Figure 7).
By their lipidic nature, these analogs might act through a detergent effect that could lead to lysis or modification of the integrity of infected-erythrocyte membranes. This apparently is not the case. No significant lysis of normal erythrocytes was observed after 48 hous of incubation with concentrations of analogs up to 10 μM (data not shown). Furthermore, no preferential lysis of infected-erythrocytes was observed on Giemsa-stained smears of infected cultures maintained 48 hours with 250 nM AD2646, a concentration inhibiting parasite growth totally.
Interestingly, the absence of a fatty acyl carbonyl group (methylene linkage) in our ceramide analogs is a critical factor for the efficacy of their antiplasmodial activity. Sphingolipids preferentially interact with cholesterol in membranes, especially in detergent-resistant microdomains (DRMs or rafts). Rafts have been described in Plasmodium and are involved, at least, in the uptake of erythrocyte raft proteins and maintenance of the parasitophorous vacuole containing the parasite, inside the erythrocyte . This interaction implies : 1) van der Waals interactions between the saturated acyl chain and sphingoid moiety of sphingolipids and the rigid planar tetracyclic rings of cholesterol  and 2) hydrogen bonds between the 3-β hydroxyl group of cholesterol and the fatty acyl carbonyl group resulting from the amide linkage with the sphingoid moiety . The amide-linked fatty acid function seems to have a profound stabilizing effect on cholesterol-sphingolipid interactions . It could be hypothesized that in a membrane context, methylene analogs might have a destabilizing effect on the cholesterol-sphingolipid interactions and, in consequence, modifications of membrane properties. Indeed, P. falciparum growth is characterized by a setting up of new permeabilities of the infected-erythrocyte membrane . Although the biochemical nature of these new permeabilities is still unknown, they have been characterized from an electrophysiological point of view and involve a malaria-induced anion channel [42, 43]. The effect of ceramide analogs was investigated on the properties of this channel. A 24 hours-incubation of infected-erythrocytes with 250 nM AD2646 or 10 μM PPMP did not modulate significantly the induced channel activity measured in the whole-cell configuration of the patch-clamp technique (S. Egee, unpublished data), suggesting that these ceramide analogs do not inhibit parasite growth through modifications of infected-erythrocyte membrane permeabilities.
Ceramide is at the parting of different ways of sphingolipid metabolism. Analogs have the potential to inhibit different ceramide-metabolizing enzymes and then might have a pleiotropic effect. Ceramide analogs in amide linkage were described as potent inhibitors of alkaline ceramidase in HL60 human myeloid leukemic cells [44, 45]. Methylene analogs inhibit the biosynthesis of SPM and glycosphingolipids in HL60 cells, and acid ceramidase in vitro . When applied to cancer cells, such analogs induced an elevation of the endogenous level of ceramide with the consequent effects of growth suppression and cell death by apoptosis [44, 45]. In contrast to what was observed for cancer cells , preliminary results suggest that the ceramide analog AD2646 induced non-apoptotic death of P. falciparum. Parasites exposed to 1 μM AD2646 for up to 36 hours failed to exhibit characteristic apoptosis, as determined by terminal deoxynucleotidyl transferase DNA fragmentation assay and DNA fragmentation using both gel electrophoresis and fluorescence microscopy methods, although the nucleus appeared highly condensed (M. Dellinger, unpublished data). Apoptosis in P. falciparum is still controversial although some characteristics of apoptosis has been described in Plasmodium . Recently, an increase in the intracellular ceramide content and an activation of parasite sphingomyelinase(s) were found to be associated with a non-apoptotic parasite death process as induced by artemisinine and mefloquine . The hypothesis that AD2646 induced parasite death through modulation of endogenous ceramide level, as observed for cancer cells, is under investigation.
This work was supported by the French Ministry of Research and New Technologies and by a grant from the Israel Science Foundation (No 607/02). We thank Prof. Joseph Schrével, Dr Isabelle Florent, Dr Stefan H.I.Kappe and Dr James Trotter for reading the manuscript and their fruitful comments.
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