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The malaria parasite, Plasmodium falciparum , develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d 9 , ethanolamine-d 4 , and serine-d 3 ) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches., (Copyright © 2018 Wein et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei -infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei -infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs., (Copyright © 2017 American Society for Microbiology.)

Severe malaria is a life-threatening complication of an infection with the protozoan parasite Plasmodium falciparum, which requires immediate treatment. Safety and efficacy concerns with currently used drugs accentuate the need for new chemotherapeutic options against severe malaria. Here we describe a medicinal chemistry program starting from amicarbalide that led to two compounds with optimized pharmacological and antiparasitic properties. SC81458 and the clinical development candidate, SC83288, are fast-acting compounds that can cure a P. falciparum infection in a humanized NOD/SCID mouse model system. Detailed preclinical pharmacokinetic and toxicological studies reveal no observable drawbacks. Ultra-deep sequencing of resistant parasites identifies the sarco/endoplasmic reticulum Ca 2+ transporting PfATP6 as a putative determinant of resistance to SC81458 and SC83288. Features, such as fast parasite killing, good safety margin, a potentially novel mode of action and a distinct chemotype support the clinical development of SC83288, as an intravenous application for the treatment of severe malaria., Competing Interests: The authors declare no competing financial interests.

Plasmodium falciparum is responsible for severe malaria which is one of the most prevalent and deadly infectious diseases in the world. The antimalarial therapeutic arsenal is hampered by the onset of resistance to all known pharmacological classes of compounds, so new drugs with novel mechanisms of action are critically needed. Albitiazolium is a clinical antimalarial candidate from a series of choline analogs designed to inhibit plasmodial phospholipid metabolism. Here we developed an original chemical proteomic approach to identify parasite proteins targeted by albitiazolium during their native interaction in living parasites. We designed a bifunctional albitiazolium-derived compound (photoactivable and clickable) to covalently crosslink drug-interacting parasite proteins in situ followed by their isolation via click chemistry reactions. Mass spectrometry analysis of drug-interacting proteins and subsequent clustering on gene ontology terms revealed parasite proteins involved in lipid metabolic activities and, interestingly, also in lipid binding, transport, and vesicular transport functions. In accordance with this, the albitiazolium-derivative was localized in the endoplasmic reticulum and trans-Golgi network of P. falciparum. Importantly, during competitive assays with albitiazolium, the binding of choline/ethanolamine phosphotransferase (the enzyme involved in the last step of phosphatidylcholine synthesis) was substantially displaced, thus confirming the efficiency of this strategy for searching albitiazolium targets.

Bis-thiazolium salts constitute a new class of antihematozoan drugs that inhibit parasite phosphatidylcholine biosynthesis. They specifically accumulate in Plasmodium- and Babesia-infected red blood cells (IRBC). Here, we provide new insight into the choline analogue albitiazolium, which is currently being clinically tested against severe malaria. Concentration-dependent accumulation in P. falciparum-infected erythrocytes reached steady state after 90 to 120 min and was massive throughout the blood cycle, with cellular accumulation ratios of up to 1,000. This could not occur through a lysosomotropic effect, and the extent did not depend on the food vacuole pH, which was the case for the weak base chloroquine. Analysis of albitiazolium accumulation in P. falciparum IRBC revealed a high-affinity component that was restricted to mature stages and suppressed by pepstatin A treatment, and thus likely related to drug accumulation in the parasite food vacuole. Albitiazolium also accumulated in a second high-capacity component present throughout the blood cycle that was likely not related to the food vacuole and also observed with Babesia divergens-infected erythrocytes. Accumulation was strictly glucose dependent, drastically inhibited by H+/K+ and Na+ ionophores upon collapse of ionic gradients, and appeared to be energized by the proton-motive force across the erythrocyte plasma membrane, indicating the importance of transport steps for this permanently charged new type of antimalarial agent. This specific, massive, and irreversible accumulation allows albitiazolium to restrict its toxicity to hematozoa-infected erythrocytes. The intraparasitic compartmentation of albitiazolium corroborates a dual mechanism of action, which could make this new type of antimalarial agent resistant to parasite resistance., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Plasmodium is the causal parasite of malaria, infectious disease responsible for the death of up to one million people each year. Glycerophospholipid and consequently membrane biosynthesis are essential for the survival of the parasite and are targeted by a new class of antimalarial drugs developed in our lab. In order to understand the highly redundant phospholipid synthethic pathways and eventual mechanism of resistance to various drugs, an organism specific kinetic model of these metabolic pathways need to be developed in Plasmodium species., Results: Fluxomic data were used to build a quantitative kinetic model of glycerophospholipid pathways in Plasmodium knowlesi. In vitro incorporation dynamics of phospholipids unravels multiple synthetic pathways. A detailed metabolic network with values of the kinetic parameters (maximum rates and Michaelis constants) has been built. In order to obtain a global search in the parameter space, we have designed a hybrid, discrete and continuous, optimization method. Discrete parameters were used to sample the cone of admissible fluxes, whereas the continuous Michaelis and maximum rates constants were obtained by local minimization of an objective function.The model was used to predict the distribution of fluxes within the network of various metabolic precursors.The quantitative analysis was used to understand eventual links between different pathways. The major source of phosphatidylcholine (PC) is the CDP-choline Kennedy pathway.In silico knock-out experiments showed comparable importance of phosphoethanolamine-N-methyltransferase (PMT) and phosphatidylethanolamine-N-methyltransferase (PEMT) for PC synthesis.The flux values indicate that, major part of serine derived phosphatidylethanolamine (PE) is formed via serine decarboxylation, whereas major part of phosphatidylserine (PS) is formed by base-exchange reactions.Sensitivity analysis of CDP-choline pathway shows that the carrier-mediated choline entry into the parasite and the phosphocholine cytidylyltransferase reaction have the largest sensitivity coefficients in this pathway, but does not distinguish a reaction as an unique rate-limiting step., Conclusion: We provide a fully parametrized kinetic model for the multiple phospholipid synthetic pathways in P. knowlesi. This model has been used to clarify the relative importance of the various reactions in these metabolic pathways. Future work extensions of this modelling strategy will serve to elucidate the regulatory mechanisms governing the development of Plasmodium during its blood stages, as well as the mechanisms of action of drugs on membrane biosynthetic pathways and eventual mechanisms of resistance.

Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-containing phospholipids, such as phosphatidylcholine (PC). According to published data, Trypanosoma brucei parasites are unable to take up choline from the environment but instead use lyso-phosphatidylcholine as precursor for choline lipid synthesis. We now show that T. brucei procyclic forms in culture readily incorporate [(3)H]-labeled choline into PC, indicating that trypanosomes express a transporter for choline at the plasma membrane. Characterization of the transport system in T. brucei procyclic and bloodstream forms shows that uptake of choline is independent of sodium and potassium ions and occurs with a Km in the low micromolar range. In addition, we demonstrate that choline uptake can be blocked by the known choline transport inhibitor, hemicholinium-3, and by synthetic choline analogs that have been established as anti-malarials. Together, our results show that T. brucei parasites express an uptake system for choline and that exogenous choline is used for PC synthesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Malaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. Several methodologies have been used to assess parasite viability during the adaption of field strains to culture or the assessment of drug potential, but these are in general not able to provide an accurate real-time assessment of whether parasites are alive or dead., Methods: Different commercial dyes and kits were assessed for their potential to allow for the real-time detection of whether a blood stage malaria parasite is dead or alive., Results: Here, a methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live (red staining) and/or dead (absence of red staining) blood stage parasites in vitro and ex vivo. This method is applicable across malaria parasite species and strains and allows to visualize all parasite blood stages including gametocytes. Further, this methodology has been assessed also for use in drug sensitivity testing., Conclusions: The JC-1 staining approach is a versatile methodology that can be used to assess parasite viability during the adaptation of field samples to culture and during drug treatment. It was found to hold promise in the assessment of drugs expected to lead to delayed death phenotypes and it currently being evaluated as a method for the assessment of parasite viability during the adaptation of patient-derived Plasmodium vivax to long-term in vitro culture.

Bis-thiazolium salts are able to inhibit phosphatidylcholine biosynthesis in Plasmodium and to block parasite proliferation in the low nanomolar range. However, due to their physicochemical properties (i.e., permanent cationic charges, the flexibility, and lipophilic character of the alkyl chain), the oral bioavailability of these compounds is low. New series of bis-thiazolium-based drugs have been designed to overcome this drawback. They feature linker rigidification via the introduction of aromatic rings and/or a decrease in the overall lipophilicity through the introduction of heteroatoms. On the basis of the structure-activity relationships, a few of the promising compounds (9, 10, and 11) were found to exhibit potent antimalarial in vitro and in vivo activities (EC(50) < 10 nM and ED(50) ip < 0.7 mg/kg).

Plasmodium falciparum is the causative agent of malaria, a deadly infectious disease for which treatments are scarce and drug-resistant parasites are now increasingly found. A comprehensive method of identifying and quantifying metabolites of this intracellular parasite could expand the arsenal of tools to understand its biology, and be used to develop new treatments against the disease. Here, we present two methods based on liquid chromatography tandem mass spectrometry for reliable measurement of water-soluble metabolites involved in phospholipid biosynthesis, as well as several other metabolites that reflect the metabolic status of the parasite including amino acids, carboxylic acids, energy-related carbohydrates, and nucleotides. A total of 35 compounds was quantified. In the first method, polar compounds were retained by hydrophilic interaction chromatography (amino column) and detected in negative mode using succinic acid-(13)C(4) and fluorovaline as internal standards. In the second method, separations were carried out using reverse phase (C18) ion-pair liquid chromatography, with heptafluorobutyric acid as a volatile ion pairing reagent in positive detection mode, using d(9)-choline and 4-aminobutanol as internal standards. Standard curves were performed in P. falciparum-infected and uninfected red blood cells using standard addition method (r(2)>0.99). The intra- and inter-day accuracy and precision as well as the extraction recovery of each compound were determined. The lower limit of quantitation varied from 50pmol to 100fmol/3×10(7)cells. These methods were validated and successfully applied to determine intracellular concentrations of metabolites from uninfected host RBCs and isolated Plasmodium parasites., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We report herein the design, synthesis, and biological screening of a series of 15 disulfide prodrugs as precursors of albitiazolium bromide (T3/SAR97276, compound 1), a choline analogue which is currently being evaluated in clinical trials (phase II) for severe malaria. The corresponding prodrugs are expected to revert back to the active bis-thiazolium salt through an enzymatic reduction of the disulfide bond. To enhance aqueous solubility of these prodrugs, an amino acid residue (valine or lysine) or a phosphate group was introduced on the thiazolium side chain. Most of the novel derivatives exhibited potent in vitro antimalarial activity against P. falciparum. After oral administration, the cyclic disulfide prodrug 8 showed the best improvement of oral efficacy in comparison to the parent drug.

Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, which has been shown to be important in various processes of the malaria parasite Plasmodium. PI-PLC is generally implicated in calcium liberation from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate, and is itself dependent on calcium for its activation. Here we describe the plc genes from Plasmodium species. The encoded proteins contain all domains typically found in PI-PLCs of the δ class but are almost twice as long as their orthologues in mammals. Transcriptional analysis by qRT-PCR of plc during the erythrocytic cycle of P. falciparum revealed steady expression levels that increased at the late schizont stages. Genetic analysis in the P. berghei model revealed that the plc locus was targetable but that plc gene knock-outs could not be obtained, thereby strongly indicating that the gene is essential during blood stage development. Alternatively, we attempted to modify plc expression through a promoter exchange approach but found the gene to be refractory to over-expression indicating that plc expression levels might additionally be tightly controlled., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In the human malaria parasite Plasmodium falciparum, the synthesis of the major and essential membrane phospholipid, phosphatidylcholine, occurs via the CDP-choline and the serine decarboxylase phosphoethanolamine methylation (SDPM) pathways, which are fueled by host choline, serine, and fatty acids. Both pathways share the final two steps catalyzed by two essential enzymes, P. falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) and choline-phosphate transferase (PfCEPT). We identified a novel class of phospholipid mimetics, which inhibit the growth of P. falciparum as well as Leishmania and Trypanosoma species. Metabolic analyses showed that one of these compounds, PG12, specifically blocks phosphatidylcholine biosynthesis from both the CDP-choline and SDPM pathways via inhibition of PfCCT. In vitro studies using recombinant PfCCT showed a dose-dependent inhibition of the enzyme by PG12. The potent antimalarial of this compound, its low cytotoxicity profile, and its established mode of action make it an excellent lead to advance for further drug development and efficacy in vivo.

Critical events in the life cycle of malaria parasites are controlled by calcium-dependent signalling cascades, yet the molecular mechanisms of calcium release remain poorly understood. The synchronized development of Plasmodium berghei gametocytes relies on rapid calcium release from internal stores within 10 s of gametocytes being exposed to mosquito-derived xanthurenic acid (XA). Here we addressed the function of phosphoinositide-specific phospholipase C (PI-PLC) for regulating gametocyte activation. XA triggered the hydrolysis of PIP(2) and the production of the secondary messenger IP(3) in gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca(2+) signal. However, microgametocyte differentiation into microgametes was blocked even when the inhibitor was added up to 5 min after activation, suggesting a requirement for PI-PLC beyond the early mobilization of calcium. In contrast, inhibitors of calcium release through ryanodine receptor channels were active only during the first minute of gametocyte activation. Biochemical determination of PI-PLC activity was confirmed using transgenic parasites expressing a fluorescent PIP(2) /IP(3) probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca(2+) and PI-PLC activity, with PI-PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process., (© 2011 Blackwell Publishing Ltd.)

Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

Antimalarial activities and pharmacokinetics of the bis-alkylamidine, M64, and its amidoxime, M64-AH, and O-methylsulfonate, M64-S-Me, derivatives were investigated. M64 and M64-S-Me had the most potent activity against the Plasmodium falciparum growth (IC(50)<12nM). The three compounds can clear the Plasmodium vinckei infection in mice (ED(50)<10mg/kg). A liquid chromatography-mass spectrometry method was validated to simultaneously quantify M64 and M64-AH in human and rat plasma. M64 is partially metabolized to M64-monoamidoxime and M64-monoacetamide by rat and mouse liver microsomes. The amidoxime M64-AH undergoes extensive metabolism forming M64, M64-monoacetamide, M64-diacetamide and M64-monoamidoxime. Strong interspecies differences were observed. The pharmacokinetic profiles of M64, M64-AH and M64-S-Me were studied in rat after intravenous and oral administrations. M64 is partially metabolized to M64-AH; while M64-S-Me is rapidly and totally converted to M64 and M64-AH. M64-AH is mostly oxidized to the inactive M64-diacetamine while its N-reduction to the efficient M64 is a minor metabolic pathway. Oral dose of M64-AH was well absorbed (38%) and converted to M64 and M64-diacetamide. This study generated substantial information about the properties of this class of antimalarial drugs. Other routes of synthesis will be explored to prevent oxidative transformation of the amidoxime and to favour the N-reduction., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objectives: to investigate the anti-kinetoplastid activity of choline-derived analogues with previously reported antimalarial efficacy., Methods: from an existing choline analogue library, seven antimalarial compounds, representative of the first-, second- and third-generation analogues previously developed, were assessed for activity against Trypanosoma and Leishmania spp. Using a variety of techniques, the effects of choline analogue exposure on the parasites were documented and a preliminary investigation of their mode of action was performed., Results: the activities of choline-derived compounds against Trypanosoma brucei and Leishmania mexicana were determined. The compounds displayed promising anti-kinetoplastid activity, particularly against T. brucei, to which 4/7 displayed submicromolar EC(50) values for the wild-type strain. Low micromolar concentrations of most compounds cleared trypanosome cultures within 24-48 h. The compounds inhibit a choline transporter in Leishmania, but their entry may not depend only on this carrier; T. b. brucei lacks a choline carrier and the mode of uptake remains unclear. The compounds had no effect on the overall lipid composition of the cells, cell cycle progression or cyclic adenosine monophosphate production or short-term effects on intracellular calcium levels. However, several of the compounds, displayed pronounced effects on the mitochondrial membrane potential; this action was not associated with production of reactive oxygen species but rather with a slow rise of intracellular calcium levels and DNA fragmentation., Conclusions: the choline analogues displayed strong activity against kinetoplastid parasites, particularly against T. b. brucei. In contrast to their antimalarial activity, they did not act on trypanosomes by disrupting choline salvage or phospholipid metabolism, instead disrupting mitochondrial function, leading to chromosomal fragmentation.

Phosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasite Plasmodium falciparum establishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile of P. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. The Plasmodium genome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted in Plasmodium berghei, and in vitro growth of P. falciparum was sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential in Plasmodium blood stages. For intraparasitic PI3P localization, transgenic P. falciparum that expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function in Plasmodium appear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.

Throughout the Plasmodium life cycle, malaria parasites repeatedly undergo rapid cellular growth and prolific divisions, necessitating intense membrane neogenesis and, in particular, the acquisition of high amounts of phospholipids. At the intraerythrocytic stage, glycerophospholipids are the main parasite membrane constituents, which mostly originate from the Plasmodium-encoded enzymatic machinery. Several proteins and entire pathways have been characterized and their features reported, thereby generating a global view of glycerophospholipid synthesis across Plasmodium spp. The malaria parasite displays a panoply of pathways that are seldom found together in a single organism. The major glycerophospholipids are synthesized via ancestral prokaryotic CDP-diacylglycerol-dependent pathways and eukaryotic-type de novo pathways. The parasite exhibits additional reactions that bridge some of these routes and are otherwise restricted to some organisms, such as plants, while base-exchange mechanisms are largely unexplored in Plasmodium. Marked differences between Plasmodium spp. have also been reported in phosphatidylcholine and phosphatidylethanolamine synthesis. Little is currently known about glycerophospholipid acquisition at non-erythrocytic stages, but recent data reveal that intrahepatocytic parasites, oocysts and sporozoites import various host lipids, and that de novo fatty acid synthesis is only crucial at the late liver stage. More studies on the different Plasmodium developmental stages are needed, to further assemble the different pieces of this glycerophospholipid synthesis puzzle, which contains highly promising therapeutic targets., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main membrane phospholipids (PLs) of Plasmodium parasites and can be generated by the de novo (Kennedy) CDP-choline and CDP-ethanolamine pathways and by the CDP-diacylglycerol dependent pathway. The Kennedy pathways initiate from exogenous choline and ethanolamine involving choline kinase (CK) and ethanolamine kinase (EK), followed by the choline-phosphate cytidylyltransferase (CCT) and ethanolamine-phosphate cytidylyltransferase (ECT) that catalyse the formation of CDP-choline and CDP-ethanolamine. Finally, in Plasmodium, PC and PE are apparently synthesized by a common choline/ethanolamine-phosphotransferase (CEPT). Here, we have studied the essential nature of the Kennedy pathways in Plasmodium berghei, a rodent malaria parasite. Sequence analysis of the P. berghei CEPT, CCT, ECT and CK enzymes revealed the presence of all catalytic domains and essential residues and motifs necessary for enzymatic activities. Constructs were designed for the generation of gene knockout and GFP-fusions of the cept, cct, ect and ck genes in P. berghei. We found that all four genes were consistently refractory to knockout attempts. At the same time, successful tagging of these proteins with GFP demonstrated that the loci were targetable and indicated that these genes are essential in P. berghei blood stage parasites. GFP-fusions of CCT, ECT and CK were found in the cytosol whereas the GFP-CEPT mainly localised in the endoplasmic reticulum. These results indicate that both CDP-choline and CDP-ethanolamine de novo pathways are essential for asexual P. berghei development and are non-redundant with other possible sources of PC and PE., (Copyright 2010 Elsevier B.V. All rights reserved.)

Cytidine diphosphate diacylglycerol synthase (CDS) diverts phosphatidic acid towards the biosynthesis of CDP-DAG, an obligatory liponucleotide intermediate in anionic phospholipid biosynthesis. The 78kDa predicted Plasmodium falciparum CDS (PfCDS) is recovered as a 50 kDa conserved C-terminal cytidylyltransferase domain (C-PfCDS) and a 28kDa fragment that corresponds to the unusually long hydrophilic asparagine-rich N-terminal extension (N-PfCDS). Here, we show that the two fragments of PfCDS are the processed forms of the 78 kDa pro-form that is encoded from a single transcript with no alternate translation start site for C-PfCDS. PfCDS, which shares 54% sequence identity with Plasmodium knowlesi CDS (PkCDS), could substitute for PkCDS in P. knowlesi. Experiments to disrupt either the full-length or the N-terminal extension of PkCDS indicate that not only the C-terminal cytidylyltransferase domain but also the N-terminal extension is essential to Plasmodium spp. PkCDS and PfCDS introduced in P. knowlesi were processed in the parasite, suggesting a conserved parasite-dependent mechanism. The N-PfCDS appears to be a peripheral membrane protein and is trafficked outside the parasite to the parasitophorous vacuole. Although the function of this unusual N-PfCDS remains enigmatic, the study here highlights features of this essential gene and its biological importance during the intra-erythrocytic cycle of the parasite., (Copyright (c) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

SUMMARYPlasmodium falciparum, the agent responsible for malaria, is an obligate intracellular protozoan parasite. For proliferation, differentiation and survival, it relies on its own protein-encoding genes, as well as its host cells for nutrient sources. Nutrients and subsequent metabolites are required by the parasites to support their high rate of growth and replication, particularly in the intra-erythrocytic stages of the parasite that are responsible for the clinical symptoms of the disease. Advances in mass spectrometry have improved the analysis of endogenous metabolites and enabled a global approach to identify the parasite's metabolites by the so-called metabolomic analyses. This level of analysis complements the genomic, transcriptomic and proteomic data already available and should allow the identification of novel metabolites, original pathways and networks of regulatory interactions within the parasite, and between the parasite and its hosts. The field of metabolomics is just in its infancy in P. falciparum, hence in this review, we concentrate on the available methodologies and their potential applications for deciphering important biochemical processes of the parasite, such as the astonishingly diverse phospholipid biosynthesis pathways. Elucidating the regulation of the biosynthesis of these crucial metabolites could help design of future anti-malarial drugs.

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.

Targeting the crucial step of Plasmodium transition from vertebrate host to mosquito vector is a promising approach to eliminate malaria. Uptake by the mosquito activates gametocytes within seconds, and in the case of male (micro) gametocytes leads to rapid DNA replication and the release of eight flagellated gametes. We developed a sensitive assay to monitor P. berghei microgametocyte activation based on [(3)H]hypoxanthine incorporation into DNA. Optimal pH range and xanthurenic acid concentrations for gametocyte activation were established and the kinetics of DNA replication investigated. Significance of the method was confirmed using P. berghei mutants and the assay was applied to analyse the effect of protease inhibitors, which revealed differences regarding their inhibitory action. The developed method thus appears suitable for reproducible determination of microgametocyte activation, medium-throughput drug screenings and deeper investigation of early blocks in gametogenesis and will facilitate the analysis of compounds for transmission blocking activities.

Since several years, we developed a new class of antimalarial drugs targeting the phospholipid metabolism of the Plasmodium falciparum malaria parasite. The bis-thiazolium compound, SAR97276, is the lead compound and is now in clinical development. In this paper, we applied the fast rapid resolution liquid chromatography-mass spectrometry technique to the analysis of SAR97276 in monkey matrices. The sample pre-treatment procedure involved an acidic precipitation of proteins followed by solid-phase extraction. The monocationic compound, T2, was used as internal standard. A good separation was achieved on a Zorbax eclipse XDB C8 column (1.8 microm, 50 mm x 4.6mm) with a mobile phase consisting of acetonitrile-trimethylamine-formate buffer (pH 3) gradient elution. The total run time was 8 min. Inter-assay precisions were <10% in plasma, and 85% in plasma, and >75% in blood. The lower limits of quantitation were 3.3 microg/l in plasma and 3.3 microg/kg in blood. No matrix effect was observed. This newly developed method is sensitive, selective, reproducible, and stability indicating. It was used to analyse samples taken during a pharmacokinetic/pharmacodynamic study carried out in infected Rhesus monkey by Plasmodium cynomolgi as part of the ongoing development of SAR97276.

1,12-Bis[5-(2-hydroxyethyl)-4-methyl-1,3-thiazol-3-ium]dodecane dibromide (SAR97276, T3) is a new antimalarial drug, which is currently being evaluated in clinical trials for severe malaria. Drug accumulation inside the parasite and a dual mechanism of action are a major strength of this compound, as it could help delay the development of resistance. The purpose of this article was to develop a rapid resolution LC-MS method for quantifying SAR97276 in mouse tissues. The LC system consisted of Zorbax Eclipse XDB C8 (1.8 microm, 50 x 4.6 mm, 60 degrees C) column. Elution with a gradient mobile phase consisting of ACN-trimethylamine-formate buffer (pH 3) at a flow rate of 1 mL/min yielded sharp, utmost-resolved peaks within 2 min. Tissue samples were powdered under liquid nitrogen. After protein precipitation with citric acid, SPE using WCX cartridges was used for sample preparation. There was no influence of the matrix on the detection of either SAR97276 or the IS. Assay precision was <13% and accuracy was 90-107%. The lower LOQs were 3.3 microg/kg in brain and 33 microg/kg in liver and heart. This newly developed method was used to study the tissue distribution of SAR97276 in mouse as part of the ongoing development of SAR97276.

Preclinical animal models informing antimalarial drug development are scarce. We have used asexual erythrocytic Plasmodium cynomolgi infections of rhesus macaques to model Plasmodium vivax during preclinical development of compounds targeting parasite phospholipid synthesis. Using this malaria model, we accumulated data confirming highly reproducible infection patterns, with self-curing parasite peaks reproducibly preceding recrudescence peaks. We applied nonlinear mixed-effect (NLME) models, estimating treatment effects in three drug studies: G25 (injected) and the bisthiazolium prodrugs TE4gt and TE3 (oral). All compounds fully cured P. cynomolgi-infected macaques, with significant effects on parasitemia height and time of peak. Although all three TE3 doses tested were fully curative, NLME models discriminated dose-dependent differential pharmacological antimalarial activity. By applying NLME modeling treatment effects are readily quantified. Such drug development studies are more informative and contribute to reduction and refinement in animal experimentation.

A new class of antimalarial drugs targeting phospholipid metabolism of the malarial parasite is now in development. In the strategy of this development, two mono-thiazolium salts, T1 and T2, need to be monitored. A liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated according to FDA guidelines for simultaneous determination of T1 and T2 in plasma, whole blood and red blood cells (RBCs) from human and rat. The sample-pre-treatment procedure involved solid phase extraction after protein precipitation. Chromatography was carried out on a Zorbax eclipse XDB C8 column and mass spectrometric analysis was performed using an Agilent 1,100 quadrupole mass spectrometer working with an electrospray ionization source. LC-MS data were acquired in single ion monitoring mode at m/z 312, 326 and 227 for T1, T2 and the internal standard (T3), respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to concentrations (human and rat plasma: 2.25-900 microg/l; human blood and rat RBCs: 4.5-900 microg/kg). Precision was below 14.5% for T1 and below 13% for T2. Accuracy was 92.6-111% for T1 and 95.6-108% for T2. Extraction recoveries were >or=85% in plasma and >or=53% in blood and RBCs. For T1 and T2, the lower limits of quantitation were 2.25 microg/l in plasma, and 4.5 microg/kg in whole blood and RBCs. Stability tests under various conditions were also investigated. This highly specific and sensitive method was useful to analyse samples from pharmacokinetic studies carried out in rat and would also be useful in clinical trials at a later stage.

A sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of a new antimalarial bisthiazolium salt, SAR97276, in mouse plasma and red blood cells (RBCs). A drug of the same chemical series as the test drug, T2, was used as internal standard. The method involved solid phase extraction of the compound and the internal standard from the two matrices using Oasis HLB columns. LC separation was performed on a Zorbax eclipse XDB C8 column (5 microm) with a mobile phase of acetonitrile containing trimethylamine (130 microl/l, solvent A) and 2 mM ammonium formate buffer (solvent B). MS data were acquired in single ion monitoring mode at m/z 227 for SAR97276 and m/z 326 for T2. The matrix had no influence on the detection of either SAR97276 or T2. The drug/internal standard peak area ratios were linked via quadratic relationships to plasma (1.65-1322 ng/ml) and RBC concentrations (3.31-2644 ng/ml). Precision was below 14% and accuracy was 91.4-104%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries of SAR97276 were > or =90% in plasma and > or =60% in RBCs. The lower limits of quantitation were 1.65 ng/ml in plasma and 3.31 ng/ml in RBCs. Stability tests under various conditions were also investigated. The method was successfully used to determine the pharmacokinetic profile of SAR97276 in healthy mouse.

Phosphatidylinositol (PI) is a versatile lipid that not only serves as a structural component of cellular membranes, but also plays important roles in membrane anchorage of proteins and in signal transduction through distinct phosphorylated derivatives of the inositol head group. PI is synthesised by PI synthase from CDP-diacylglycerol and myo-inositol. The enzymatic activity in Plasmodium falciparum and P. knowlesi has previously been characterised at the biochemical level. Here we characterise the PI synthase gene of P. falciparum and P. knowlesi. The cDNA sequence identified a highly spliced gene consisting of nine exons and encoding a protein of 209 and 207 amino acids, respectively. High sequence conservation enabled the prediction of the PI synthase genes of P. berghei, P. chabaudi and P. vivax. All Plasmodium PI synthase proteins appear to be highly hydrophobic, although no consensus for the number and location of distinct transmembrane domains could be detected. The P. falciparum PI synthase (PfPIS) gene successfully complemented a Saccharomyces cerevisiae PIS1 deletion mutant, demonstrating its enzymatic function. Complementation efficiency was dramatically improved when hybrid constructs between N-terminal S. cerevisiae and C-terminal P. falciparum sequences were used. Determination of in vitro PIS activities of complemented yeast strains confirmed the enzymatic function of the Plasmodium protein.

A leading bisthiazolium drug, T16, designed to mimic choline, was shown to exert potent antibabesial activity, with 50% inhibitory concentrations of 28 and 7 nM against Babesia divergens and B. canis, respectively. T16 accumulated inside Babesia-infected erythrocytes (cellular accumulation ratio, >60) by a saturable process with an apparent K(m) of 0.65 microM. Subcellular fractionation of Babesia parasites revealed the accumulation of T16 into a low-density fraction, while in malaria-infected erythrocytes a significant fraction of the drug was associated with heme malaria pigment. T16 exerts an early and specific inhibition of the de novo biosynthesis of phosphatidylcholine both in B. divergens- and Plasmodium falciparum-infected erythrocytes. Choline accumulation into isolated Babesia parasites was highly sensitive to inhibition by T16. These data are consistent with the hypothesis that bisthiazolium drugs target the de novo phosphatidylcholine biosynthesis of intraerythrocytic hematozoan parasites. In malaria parasites, which generate ferriprotoporphyrin IX during hemoglobin digestion, T16 binding to heme may enhance the accumulation and activity of the drug. The selectivity of accumulation and potent activity of this class of drug into parasite-infected erythrocytes offers unique advantages over more traditional antihematozoan drugs.

Babesiosis is caused by a haemotropic protozoal parasite of the genus Babesia, member of the phylum Apicomplexa and transmitted by the bite of an infected tick. There are many Babesia species affecting livestock, dogs, horses and rodents which are of economic significance. Infections can occur without producing symptoms, but babesiosis may also be severe and sometimes fatal caused by the intraerythrocytic parasite development. The disease can cause fever, fatigue and haemolytic anemia lasting from several days to several months. There are a number of effective babesiacides, but imidocarb dipropionate (which consistently clears the parasitaemia; often the only available drug on the market) and diminazene aceturate are the most widely used. Some Babesia spp. can infect humans, particularly Babesia microti and Babesia divergens, and human babesiosis is a significant emerging tick-borne zoonotic disease. Clinical manifestations differ markedly between European and North American diseases. In clinical cases, a combination of clindamycin and quinine is administered as the standard treatment, but also administration of atovaquone-azithromycin is successful. Supportive therapy such as intravenous fluids and blood transfusions are employed when necessary. More specific fast-acting new treatments for babesiosis have now to be developed. This should be facilitated by the knowledge of the Babesia spp. genome and increased interest for this malaria-like parasite.

A new approach to malarial chemotherapy based on quaternary ammonium that targets membrane biogenesis during intraerythrocytic Plasmodium falciparum development has recently been developed. To increase the bioavailability, nonionic chemically modified prodrugs were synthesized. In this paper, the pharmacological properties of a bisthiazolium salt (T3) and its bioprecursor (TE3) were studied. Their antimalarial activities were determined in vitro against the growth of P. falciparum and in vivo against the growth of P. vinckei in mice. Pharmacokinetic evaluations were performed after T3 (1.3 and 3 mg/kg of body weight administered intravenously; 6.4 mg/kg administered intraperitoneally) and TE3 (1.5 and 3 mg/kg administered intravenously; 12 mg/kg administered orally) administrations to rats. After intraperitoneal administration, very low doses offer protection in a murine model of malaria (50% efficient dose [ED50] of 0.2 to 0.25 mg/kg). After oral administration, the ED50 values were 13 and 5 mg/kg for T3 and TE3, respectively. Both compounds exerted antimalarial activity in the low nanomolar range. After TE3 administration, rapid prodrug-drug conversion occurred; the mean values of the pharmacokinetic parameters for T3 were as follows: total clearance, 1 liter/h/kg; steady-state volume of distribution, 14.8 liters/kg; and elimination half-life, 12 h. After intravenous administration, T3 plasma concentrations increased in proportion to the dose. The absolute bioavailability was 72% after intraperitoneal administration (T3); it was 15% after oral administration (TE3). T3 plasma concentrations (8 nM) 24 h following oral administration of TE3 were higher than the 50% inhibitory concentrations for the most chloroquine-resistant strains of P. falciparum (6.3 nM).

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.

Background: A new class of antimalarial drugs targeting membrane biogenesis during intraerythrocytic Plasmodium falciparum development has been identified. The bisthiazolium salts T3 and T4 have superior in vitro and in vivo parasite-killing properties and need to be monitored., Methods: We used a liquid chromatography-electrospray ionization mass spectrometry method (positive mode) to quantify two bisthiazolium compounds (T3 and T4) and a related prodrug (TE4c) in human and rat plasma. Verapamil was used as internal standard. Verapamil and the TE4c compound were characterized by protonated molecules at m/z 455.7 and m/z 725.7, respectively. T3 and T4 were detected through two ions [(M2+)/2] at m/z 227.7 and m/z 241.8 and by their adducts with trifluoroacetic acid [M+TFA]+ at m/z 568 and m/z 596, respectively. The sample clean-up procedure involved solid-phase extraction. HPLC separation was performed on a reversed-phase column, using a water-acetonitrile gradient, with both solvents containing TFA. Stability under various conditions was also investigated., Results: The peak-area ratios (drugs/internal standard) were linked to concentrations (6.4-1282 microg/L for T3; 6.5-1309.8 microg/L for T4; 20-2000 microg/L for TE4c) according to a quadratic equation. The accuracy ranged from 85% to 113.1%, and the imprecision from 2.2% to 15%. The mean extraction recoveries were 87%, 98%, and 80% for T3, T4, and TE4c, respectively. The lower limit of quantification was 6.4 mug/L for the two bisthiazolium compounds, whereas it was 20 mug/L for TE4c, the related lipophilic prodrug., Conclusion: This highly specific and sensitive method is suitable for analyzing samples collected during preclinical pharmacokinetic studies in rats and to determine the percentage binding of T3 and T4 to human plasma proteins.

A new antimalarial pharmacological approach based on inhibition of the plasmodial phospholipid metabolism has been developed. The drugs mimic choline structure and inhibit de novo phosphatidylcholine biosynthesis. Three generations of compounds were rationally designed. Bisquaternary ammonium salts showed powerful antimalarial activity, with IC(50) in the nanomolar range. To remedy their low per os absorption, bioisosteric analogues (bis-amidines) were designed and exhibited similar powerful activities. Finally, the third generation compounds are bis-thiazolium salts and their non-ionic precursors: prodrugs, which in vivo can lead to thiazolium drugs after enzymatic transformation. The compounds are equally effective against multiresistant Plasmodium falciparum malaria. These molecules exert a very rapid cytotoxic effect against malarial parasites in the very low nanomolar range and are active in vivo against P. vinckei-infected mice, with ED(50) lower than 0.2 mg/kg. They are able to cure highly infected mice and, retain full activity after a single injection. They also retain full activity against P. falciparum and P. cynomolgi in primate models with no recrudescence and at lower doses. Compounds are accumulated in P.falciparum-infected erythrocyte, which ensures their potency and specificity. Recently, we discovered that compounds also interact with malarial pigment enhancing the antimalarial effect. It is quite likely that they are dual molecules, exerting their antimalarial activity via two simultaneous toxic effects on the intracellular intraerythrocytic parasites. The current leader compounds are accessible in few steps from commercial products. These crystalline molecules present a remarkable biological activity and low toxicity which is promising for the development of a new antimalarial drug.

New drugs are urgently needed to combat the growing problem of drug resistance in Plasmodium falciparum malaria. The infected erythrocyte is a multicompartmental system, and its transporters are of interest as drug targets in their own right and as potential routes for the delivery of antimalarial drugs. Choline is an important nutrient that penetrates infected erythrocyte membranes through the endogenous carrier and through parasite-induced permeability pathways, but nothing is known about its transport into the intracellular parasite. Here we present the first characterization of choline transport across the parasite membrane. Transport exhibits Michaelis-Menten kinetics with an apparent K(m) of 25.0 +/- 3.5 muM for choline. The carrier is inhibitor-sensitive, temperature-dependent, and Na(+)-independent, and it is driven by the proton-motive force. Highly active bis-amidine and bis-quaternary ammonium compounds are also known to penetrate the host erythrocyte membrane through parasite-induced permeability pathways. Here, we demonstrate that the parasite choline transporter mediates the delivery of these compounds to the intracellular parasite. Thus, the induced permeability pathways in the host erythrocyte membrane and the parasite choline transporter described here form a cooperative transport system that shows great promise for the selective targeting of new agents for the chemotherapy of malaria.

We created neutral antimalarial prodrugs that deliver bisthiazolium compounds with antimalarial activity in the nanomolar range. These drugs primarily affect early intraerythrocytic stages through rapid, nonreversible cytotoxicity. The compounds are suitable for both parenteral and oral use and plasma promotes rapid conversion of the prodrug into the drug. We demonstrate that very low doses offer protection in a murine model of malaria. The drugs show great potential for curing high parasitemia with short-course treatments. Oral administration of the TE3 prodrug completely cures Plasmodium cynomolgi infection in rhesus monkeys. The drugs specifically accumulate inside infected erythrocytes, block phosphatidylcholine biosynthesis, and interact with hemozoin. To our knowledge, this class of compounds represents one of the most potent antimalarials tested to date. These unique properties signal a promising future for this class of antimalarial.

We characterized the potent in vitro antimalarial activity and biologic assessment of 13 phospholipid polar head analogs on a comparative basis. There was a positive relationship between the abilities of the drugs to inhibit parasite growth in culture and their abilities to specifically inhibit phosphatidylcholine biosynthesis of Plasmodium falciparum-infected erythrocytes. Maximal activity of G25 was observed for the trophozoite stage of the 48-h erythrocytic cycle (50% inhibitory concentration, 0.75 nM), whereas the schizont and ring stages were 12- and 213-fold less susceptible. The compounds exerted a rapid nonreversible cytotoxic effect, with complete clearance of parasitemia after 5 h of contact with the mature stages. The compounds were highly specific against P. falciparum, with much lower toxicity against three other mammalian cell lines, and the in vitro therapeutic indices ranged from 300 to 2,500,000. Finally, the monoquaternary ammonium E10 and two bis-ammonium salts, G5 and G25, were similarly active against multiresistant strains and fresh isolates of P. falciparum. This impressive selective in vitro toxicity against P. falciparum strongly highlights the clinical potential of these quaternary ammonium salts for malarial chemotherapy.

We previously showed that quaternary ammonium salts have potent antimalarial activities against the blood stage of drug-resistant Plasmodium falciparum. In the present study, 13 compounds of this series were comparatively assessed in murine in vivo malarial models. Mice infected with Plasmodium berghei were successfully treated with 11 quaternary ammonium salts in a 4-day suppressive test with a once-daily intraperitoneal administration. The dose required to decrease parasitemia by 50% (ED(50)) ranged from 0.04 to 4.5 mg/kg of body weight. For six mono- and three bis-quaternary ammonium salts, the therapeutic indices (i.e., 50% lethal dose and ED(50)) were higher than 5, and at best, around 20 to 30 for five of them (E6, E8, F4, G5, and G25), which is comparable to that of chloroquine under the same conditions. Plasmodium chabaudi was significantly more susceptible to G5, G15, and G25 compounds than P. berghei. Similar therapeutic indices were obtained, regardless of the administration mode or initial parasitemia (up to 11.2%). Parasitemia clearance was complete without recrudescence. Subcutaneously administered radioactive compounds had a short elimination half-life in mice (3.5 h) with low bioavailability (17.3%), which was likely due to the permanent cationic charge of the molecule. The high in vivo therapeutic index in the P. chabaudi-infected mouse model and the absence of recrudescence highlight the enormous potential of these quaternary ammonium salts for clinical malarial treatment.

Parasitic protozoa are surrounded by membrane structures that have a different lipid and protein composition relative to membranes of the host. The parasite membranes are essential structurally and also for parasite specific processes, like host cell invasion, nutrient acquisition or protection against the host immune system. Furthermore, intracellular parasites can modulate membranes of their host, and trafficking of membrane components occurs between host membranes and those of the intracellular parasite. Phospholipids are major membrane components and, although many parasites scavenge these phospholipids from their host, most parasites also synthesise phospholipids de novo, or modify a large part of the scavenged phospholipids. It was recently shown that some parasites like Plasmodium have unique phospholipid metabolic pathways. This review will focus on new developments in research on phospholipid metabolism of parasitic protozoa in relation to parasite-specific membrane structures and function, as well as on several targets for interference with the parasite phospholipid metabolism with a view to developing new anti-parasitic drugs., (Copyright 2002 Elsevier Science B.V.)

CDP-diacylglycerol synthase (CDS) is a key rate-limiting enzyme in the phospholipid metabolism of Plasmodium falciparum, converting phosphatidic acid to CDP-diacylglycerol. The CDS gene is predominantly expressed in the mature intraerythrocytic stages. Consequently, we physically and functionally characterized the CDS gene promoter. The mRNA transcription initiation site was mapped 121 bp upstream of the CDS gene translation start site. A 1909 bp 5' upstream sequence was isolated and found to be transcriptionally active thus constituting a functional CDS promoter. Mapping of this promoter identified a 44 bp cis-acting sequence, located between -1640 and -1596 bp upstream of the ATG codon, essential for efficient transcriptional activity. This 44 bp sequence binds specifically to nuclear factors from trophozoite stage parasites. We further showed that a 24 bp element, lying within the 44 bp sequence, mediates the specific binding to nuclear proteins and shows no significant homology to known eukaryotic DNA consensus sequence elements that bind transcription factors. The deletion of the 24 bp element abrogated promoter activity, indicating that this cis-acting sequence element is essential for efficient transcription of the CDS gene.

During asexual development within erythrocytes, malaria parasites synthesize considerable amounts of membrane. This activity provides an attractive target for chemotherapy because it is absent from mature erythrocytes. We found that compounds that inhibit phosphatidylcholine biosynthesis de novo from choline were potent antimalarial drugs. The lead compound, G25, potently inhibited in vitro growth of the human malaria parasites Plasmodium falciparum and P. vivax and was 1000-fold less toxic to mammalian cell lines. A radioactive derivative specifically accumulated in infected erythrocytes to levels several hundredfold higher than in the surrounding medium, and very low dose G25 therapy completely cured monkeys infected with P. falciparum and P. cynomolgi.

Carboxylic true ionophores were previously demonstrated to have efficient antimalarial activity against the human parasite Plasmodium falciparum, with a 50% inhibitory concentration around nM and generally high selectivity as compared to their toxic effects against mammalian cell lines. The decreased molecular packing of the erythrocyte membrane outer leaflet after malarial infection could explain the preferential ionophore interaction with infected erythrocytes. Monolayer penetration experiments using different phospholipid films showed strong incorporation of true carboxylic ionophores, from classes 1 (nigericin) and 2 (lasalocid), up to a surface pressure close to film collapse. The interaction was slightly higher with PC (phosphatidylcholine) monolayers than with monolayers composed of cholesterol-containing total lipid extracts from either malaria-infected or normal erythrocytes, and the two latter induced identical interactions with 5-bromo lasalocid. Surface pressure-area isotherms for pure ionophores on water and surface tension of ionophore aqueous solutions clearly highlighted the surface-active characteristics of these ionophores and allowed determination of their molecular area in compact monolayers. The estimated ionophore concentration in the mixed interfacial layers indicates that higher amounts (threefold more) of ionophores might be integrated in infected erythrocyte membrane due to their impaired molecular packing as compared to normal erythrocytes. This infection-enhanced penetration efficiency does not appear directly related to the change in erythrocyte membrane lipid composition, but it could be the basis of ionophore selectivity for infected erythrocytes. Copyright 1999 Academic Press.

Phospholipid biosynthesis in Plasmodium is of crucial importance considering the high degree of membrane biogenesis. In the de novo phosphatidylcholine pathway, the major plasmodial phospholipid, choline, first enters infected erythrocytes by a transport-mediated process, whose main kinetic characteristics are the same as in normal cells except for a considerable increase in Vm. The kinetic and functional characterizations of the choline carrier (affinity, specificity, stereoselectivity, asymmetric cyclic model, ionic dependence, limiting step in carrier translocation) have now been done, although there is no information concerning its nature and structure, despite the fact that it is likely an outstanding pharmacological target. Other unanswered questions concern the mechanisms for choline entry into the parasite. The intense lipid trafficking between the intracellular parasite and the host cell membrane also indicates that Plasmodium controls its own lipid composition as well as that of its host cell. Organelles that house the machinery for lipid synthesis, and mechanisms for trafficking and sorting, have not yet been described because of the lack of appropriate tools, but they could address fundamental questions in the contemporary cell biology of this parasite.