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Active lipid metabolism is a key process required for the intra-erythrocytic development of the malaria parasite, Plasmodium falciparum. Enzymes that hydrolyze host-derived lipids play key roles in parasite growth, virulence, differentiation, cell-signaling and hemozoin formation. Therefore, investigating enzymes involved in lipid degradation could uncover novel drug targets. We have identified in P. falciparum, a glycerophosphodiester phosphodiesterase (PfGDPD), involved in the downstream pathway of phosphatidylcholine degradation. PfGDPD hydrolyzes deacylated phospholipids, glycerophosphodiesters to glycerol-3-phosphate and choline. In this study, we have characterized PfGDPD using bioinformatics, biochemical and genetic approaches. Knockout experiments showed a requirement for PfGDPD for parasite survival. Sequence analysis revealed PfGDPD possesses the unique GDPD insertion domain sharing a cluster of conserved residues present in other GDPD homologues. We generated yellow fluorescent fusion proteins that revealed a complex distribution of PfGDPD within the parasite cytosol, parasitophorous vacuole and food vacuole. To gain insight into the role of PfGDPD, sub-cellular localization was modulated and resulted in a shift in protein distribution, which elicited no growth phenotype. Kinetic analyses suggest PfGDPD activity is Mg₂⁺ dependent and catalytically efficient at the neutral pH environment of the parasitophorous vacuole. Next, our aim was to determine the upstream pathway that provides deacylated glycerophosphodiesters as substrate for PfGDPD. We identified via bioinformatics, a P. falciparum lysophospholipase (PfLPL1) that directly generates the substrate. Knockout clones were generated and genotyped by Southern and PCR analysis. The effects of PfLPL1 knockouts on parasite fitness were studied, and the results showed that PfLPL1was not required for parasite survival and proliferation. / Ph. D.

The emergence of pesticide-resistant mosquitoes and drug-resistant parasites in the last twenty years has made control of malaria more difficult. One novel strategy to better control malaria is the development and release of transgenic mosquitoes whose enhanced immunity prevents transmission of the parasite to the mammalian host. One candidate effector gene is Anopheles stephensi nitric oxide synthase (AsNOS), whose inducible expression and subsequent synthesis of nitric oxide (NO) limits Plasmodium development in A. stephensi. In mammals, one of the most potent physiological regulators of NOS gene expression and catalytic activity is transforming growth factor-β (TGF-β). Moreover, human TGF-β can activate Drosophila melanogaster Smads, the proteins responsible for TGF-β signal transduction. We have determined that following a bloodmeal, active human TGF-β1 (hTGF-β1) persists in the midgut of A. stephensi for up to 48 hours. My data demonstrate that the midgut epithelium recognizes hTGF-β1 as an immunomodulatory cytokine. Specifically, induction of AsNOS by hTGF-β1 occurs in the midgut within minutes of bloodfeeding. Moreover, hTGF-β1 limits development of the human malaria parasite Plasmodium falciparum in the midgut. In other experiments, provision of the AsNOS catalytic inhibitor L-NAME partially reverses the effect of hTGF-β1 on Plasmodium development. These results suggest that AsNOS is a target of hTGF-β1 signaling and additional effectors that impact parasite development may be regulated by hTGF-β1 as well. The fact that hTGF-β1 signals mosquito cells to limit malaria parasite development suggests that there is an endogenous TGF-β signaling network in place. An analysis of the A. gambiae genome database revealed the presence of six TGF-β ligands, including gene duplication in the 60A gene, the first evidence of ligand gene duplication outside of chordates. In addition to five receptors, three Smads were identified in the A. gambiae genome predicted to support TGF-β/Activin- and BMP-like signaling. Midgut epithelial cells and an immunocompetent A. stephensi cell line express all three Smads, confirming that a signaling pathway is in place to support signaling by divergent exogenous and endogenous TGF-β superfamily proteins. The results presented here provide the first evidence of immunological crosstalk between divergent free living hosts of a single parasite. Further, these results imply that the interface between mammals and the mosquitoes that feed on them provide a unique opportunity for circulating molecules in the blood, including TGF-β and other cytokines, to alter the mosquito immune response. / Master of Science

Malaria continues to be a global health burden that affects millions of people worldwide each year. Increasing demand for malaria control and eradication has led research to focus on sexual development of the malaria parasite. Sexual development is initiated when pre-destined intraerythrocytic ring stage parasites leave asexual reproduction and develop into gametocytes. A mosquito vector will ingest mature gametocytes during a blood meal. Sexual reproduction will occur in the midgut, leading to the production of sporozoites that will migrate to the salivary gland. The sporozoites will be injected to another human host during the next blood meal consequently, transmitting malaria. Due to decreased drug susceptibility of mature gametocytes, more investigation of the biology and metabolic requirements of malaria parasites during gametocytogenesis, as well as during the mosquito stages, are urgently needed to reveal novel targets for development of transmission-blocking agents. Furthermore, increasing drug resistance of the parasites to current antimalarials, including slowed clearance rates to artemisinin, requires the discovery of innovative drugs against asexual intraerythrocytic stages with novel mechanisms of action. Here, we have investigated the role of the apicoplast during Plasmodium falciparum gametocytogenesis. In addition, we describe drug-screening studies that have elucidated a novel mode of action of one compound from the Malaria Box, as well as identified new natural product compounds that may be serve as starting molecules for antimalarial development. / Ph. D.

No / The indoloquinoline alkaloid cryptolepine 1 has potent in vitro antiplasmodial activity, but it is also a DNA intercalator with cytotoxic properties. We have shown that the antiplasmodial mechanism of 1 is likely to be due, at least in part, to a chloroquine-like action that does not depend on intercalation into DNA. A number of substituted analogues of 1 have been prepared that have potent activities against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and also have in common with chloroquine the inhibition of ß-hematin formation in a cell-free system. Several compounds also displayed activity against Plasmodium berghei in mice, the most potent being 2,7-dibromocryptolepine 8, which suppressed parasitemia by 89% as compared to untreated infected controls at a dose of 12.5 mg kg-1 day-1 ip. No correlation was observed between in vitro cytotoxicity and the effect of compounds on the melting point of DNA (¿Tm value) or toxicity in the mouse¿malaria model.

No / Cassia siamea L. (Fabaceae) was identified from the southwest Nigerian ethnobotany as a remedy for febrile illness. This led to the bioassay-guided fractionation of stem bark of the plant extract, using the parasite lactate dehydrogenase assay and multi-resistant strain of Plasmodium falciparum (K1) for assessing the in vitro antimalarial activity. Emodin and lupeol were isolated from the ethyl acetate fraction by a combination of chromatographic techniques. The structures of the compounds were determined by spectroscopy, co-spotting with authentic samples and comparison with literature data. Both compounds were found to be the active principles responsible for the antiplasmodial property with IC50 values of 5 µg/mL, respectively.

No / The aim of this chapter is to illustrate some current developments in natural product-derived antimalarial drugs. Traditional medicines have provided two of our most important antimalarial drugs (quinine and artemisinin) and have the potential to provide many novel antimalarial lead compounds of which several examples will be discussed. In addition, well- known natural antimalarials such as artemisinin continue to be an important focus of research and there is also increasing interest in investigating natural product sources that have not been traditionally used as antimalarials such as marine species of plants and animals. Assays based on specific malaria parasite targets such as thioredoxin reductase and heat shock protein have been employed to screen extracts and/or compounds and these have resulted in the identification of a number of potentially interesting antiplasmodial agents. However, since many victims of malaria are unable to afford antimalarial drugs, another approach adopted by some charities/NGO’s is to encourage people to grow their own medicinal plants such as Artemisia annua; some recent studies on this theme will be discussed.

Yes / Background: Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combinations continues. Methods: The resazurin-based assay was employed in evaluating the gametocytocidal properties of C. sanguinolenta and cryptolepine against the late stage (IV/V) gametocytes of Plasmodium falciparum (NF54). A fixed ratio method based on the SYBR Green I fluorescence-based assay was used to build isobolograms from a combination of cryptolepine with four standard anti-malarial drugs in vitro using the chloroquine sensitive strain 3D7. Results: Cryptolepis sanguinolenta ( IC50 = 49.65 nM) and its major alkaloid, cryptolepine ( IC50 = 1965 nM), showed high inhibitory activity against the late stage gametocytes of P. falciparum (NF54). In the interaction assays in asexual stage, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of 1.017 ± 0.06 and 1.465 ± 0.17, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant concentration ratios showed a synergistic effect (mean ΣFIC50 = 0.287 ± 0.10) whereas an antagonistic activity (mean ΣFIC50 = 4.182 ± 0.99) was seen with mefloquine. Conclusions: The findings of this study shed light on the high gametocytocidal properties of C. sanguinolenta and cryptolepine attributing their potent anti-malarial activity mainly to their effect on both the sexual and asexual stages of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose combinations.

No / Alangium lamarkii is traditionally used in India for the treatment of malaria. Partial activity guided-fractionation of the basic chloroform fraction of the methanolic extract of the bark led to the isolation of a novel tubulosine analogue, 10-demethyl-9ˊ-N-methyltubulosine (1), for which the trivial name tubulosatine is suggested. In addition, the known A. lamarkii constituents, tubulosine (2), cephaeline and emetine were isolated as potent antiplasmodial and cytotoxic constituents, but 1 was up to 1000-fold less potent than the former alkaloids against both malaria parasites and human cancer cell lines. The compounds were active against both chloroquine-sensitive and chloroquine-resistant malaria parasites, but no selective toxicity was observed towards malaria parasites compared with cancer cells with any of the alkaloids. Further work to explore the basis for the relatively weak biological activities of 1 would be worthwhile. Betulinic acid, stigmasterol and its 3-O-glucoside were isolated from the neutral chloroform fraction of the methanolic extract. / ACU, UK (Commonwealth Scholarship, Ref. No.: INCS-2004-144).