Biochemistry and Pharmacology - Theses
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ItemMechanism of action of artemisinin antimalarials and implications for drug resistance in Plasmodium falciparum( 2015)The most deadly cases of malaria in humans are caused by Plasmodium falciparum. Artemisinin-based combination therapy is the current first-line treatment against this disease. However, artemisinin resistance has recently emerged in Southeast Asia, manifesting as delayed parasite clearance times in patients. Efforts to monitor and contain artemisinin resistance were initially frustrated by a lack of correlation between in vitro parasite sensitivity to artemisinins and in vivo drug efficacy in patients. Even though artemisinins have short in vivo half-lives, standard in vitro assays have traditionally employed extended drug exposure formats to assess parasite sensitivity to the drugs. We hypothesized that short drug pulses would better mimic in vivo conditions and be more clinically relevant for artemisinins. Using novel pulsed drug exposure assays, we demonstrate that laboratory and field (Pailin, Cambodia) parasite strains exhibit stage- and strain-dependent differences in drug sensitivities. Three stages with distinct drug sensitivities are identified in laboratory strains, namely hypersensitive early rings, insensitive mid-rings and sensitive trophozoites. Moreover, using this assay format, we are able to clearly distinguish the in vitro response of sensitive and resistant Pailin strains. We find that resistant field strains exhibit the highest levels of resistance at the very early ring stage and the late schizont stage. Our detailed analysis of field strains reveals that exposure to short pulses of artemisinins induces growth retardation in both sensitive and resistant parasites. Following this growth retardation, resistant strains survive, while sensitive parasites succumb. The data suggest that artemisinins are activated, and cause cellular damage, in both strains, but resistant parasites are better able to withstand the damage. As arrest in growth is often part of a cellular stress response, we postulated that artemisinin resistance is caused by an up-regulated parasite cellular defense mechanism. Consistent with this, we demonstrate that proteasome inhibitors effectively synergize artemisinin activity against both sensitive and resistance strains, with particularly strong synergism evident during the most resistant stage of the resistant strains. This suggests that the parasite proteasome system could be targeted to enhance drug action, offering a way to overcome artemisinin-resistant malaria. The mechanism of drug action is also investigated. Trophozoites take up and digest large amounts of host hemoglobin, and previous reports showed that the products of hemoglobin digestion can act as artemisinin activators in the trophozoite stage. However, the relevant activator or mechanism of action in rings is not clear. We show that chelating the labile iron pool has little effect on artemisinin activity against early rings. By contrast, hemoglobinases inhibitors strongly antagonize artemisinin action. We show for the first time that the hemoglobinases, falcipain-2 and -3, are expressed in rings. Moreover knockout of falcipain-2 and knockdown of falcipain-3, render the very early ring stage insensitive to artemisinins. These data lead to the surprising conclusion that hemoglobin digestion is active at the early ring stage and is involved in artemisinin activation.
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ItemSynthesis and investigation of activities and modes of action of antimalarial compounds( 2015)Syntheses, structure-activity relationships and mode of action investigations of a novel compound series of conjugated quinoline-indole, revealed that they are active against blood stages of multiple strains of Plasmodium falciparum, and appear to dissipate mitochondrial potential. To aid in the study of current antimalarial drug, artemisinin, components of a “bait-and-probe” strategy were synthesised (using click chemistry). The work in this thesis has shown that the azide-linked drug-“bait” can be covalently incorporated into a protein, which acts as a potential drug target, prior to being coupled with its identifier “probe” (e.g. biotin).
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ItemDissecting the molecular basis of malaria parasite movement and host cell traversal in the mosquito midgut( 2015)Understanding the processes by which vector-borne pathogens colonise their invertebrate host is a fundamental question both in terms of the co-evolutionary biology of host-pathogen interactions and in dissecting the molecular basis of disease transmission. For the malaria parasites in particular, from the genus Plasmodium, the process by which the parasite targets and traverses the mosquito midgut epithelium is a critical bottleneck in lifecycle progression. Motility is a fundamental part of cellular life and survival for Plasmodium parasites. The motile life cycle forms achieve motility, called gliding, via the activity of an internal actomyosin motor. Although gliding is based on the well-studied system of actin and myosin, its core biomechanics are not completely understood. Although decades of research have revealed several key molecules involved in parasite traversal there is still little real understanding of the stepwise events that govern the journey of a parasite from the blood bolus to its destination under the basal lamina of the midgut. Despite the recent gains in reducing the burden of malaria disease in human populations there is still a pressing need to generate new therapeutics and strategies targeting this global pathogen. It has been recognised that any successful programme aiming towards disease eradication or elimination cannot rely on preventative and therapeutic treatments alone, but must also incorporate strategies to block parasite transmission through the mosquito vector. Much of the current literature surrounding Plasmodium transmission focuses on other lifecycle stages, neglecting the insect stage ookinete, which in itself presents a natural lifecycle bottleneck during progression through the mosquito midgut. Our findings suggest that ookinetes require dynamic actin in order to move and that this movement occurs in a left-handed helical fashion due to parasite shape. Extending our knowledge of key traversal proteins and their function, work on the vaccine candidate Cell Traversal protein for Ookinetes and Sporozoites, show that this protein is secreted in a Calcium stimulated, cGMP dependent protein kinase supported manner. Taken together, this body of work sheds light into the major interactions between the parasite and the mosquito in order to help support the broader goal to identify targets for transmission- blocking vaccine therapies against malaria disease.