Date of Award

Summer 2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Committee Director

Roland A. Cooper

Committee Member

Lesley Greene

Committee Member

Wayne Hynes

Committee Member

Robert Ratzlaff

Committee Member

Daniel Sonenshine

Abstract

Malaria is among the most debilitating diseases of man. The protozoan parasite, Plasmodium falciparum, causes over a million annual fatalities. The antimalarial trioxanes, exemplified by artemisinin, are among the few pharmaceuticals for which clinical resistance has not become widespread. Artemisinin is a naturally occurring sesquiterpene lactone, containing a unique endoperoxide pharmacophore. Despite extensive study, the precise antimalarial mechanism of action of trioxanes remains elusive. Heme iron-mediated cleavage of the endoperoxide within the parasite digestive vacuole is hypothesized to generate cytotoxic metabolites capable of alkylating heme and damaging cellular macromolecules. The hypothesis of this research is that the endoperoxide pharmacophore underlies the intracellular distribution of trioxanes; thereby identifying cellular targets of the artemisinin-based antimalarials. We therefore identified trioxane cellular accumulation patterns within laboratory cultures of P. falciparum using microscopic visualization of fluorescently-labeled artemisinin derivatives. We further determined the ability of artemisinin to generate reactive oxygen species in vitro and in vivo.

Microscopic imaging of a fluorescent trioxane-CH2CH2 O-dansyl demonstrated rapid and specific accumulation of drug within neutral lipid bodies. Fluorescent labeling was competitive with artemisinin. Experiments with inactive artemisinin derivatives and the iron chelator, desferrioxamine, demonstrated that localization within lipid bodies was both iron- and endoperoxide-dependent. An oxidation-sensitive BODIPY lipid probe showed artemisinin-mediated peroxyl radical formation in vivo that was inhibited by lipid body depletion. Moreover, lipid extracts from artemisinin-exposed parasites contained increased fatty acids and a cholesteryl ester. in vitro reactions of artemisinin and heme in the presence of digestive vacuole-associated lipids generated fluorescent peroxidation end-products whose formation was inhibited by α-tocopherol.

We found that novel, fluorescently-labeled artemisinin derivatives actively localize to digestive vacuole-associated neutral lipid bodies in P. falciparum. Further, trioxanes altered parasite lipid composition and therefore may be crucial to their antiparasitic mechanism.

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DOI

10.25777/kgfv-pk11

ISBN

9781109368888

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