Abstract :
[en] Malaria is one of the deadliest parasitic diseases that still plagues humanity in recent times. Despite being target of several eradication campaigns, the widespread presence of the multiple agents that cause this parasitosis, along with its increased adaptability into developing resistance to treatments, make this old ailment a challenge to modern medicine. Natural products with potential antiplasmodial activity are valuable to discover new therapeutic targets and shed light on new scaffolds that can be optimized into revolutionary treatments. In this thesis, metabolomics, a sensitive and robust approach that can describe the metabolites of the malaria parasite maintained in culture, was used to profile the effects of natural products on the parasite and hypothesize their mode of action. Before this could be done, workflow investigation and protocol optimization were carried out, including a study on metabolic extraction methods with state-of-the-art statistical algorithms. Further exploration revealed that time and complexity are detrimental to repeatability and robustness of the results, so a method with a single washing step followed by methanolic 90% extraction was used and analyzed through 1HNMR and/or LC-MS, depending on sample availability.
Five individual cases were studied in collaboration with the Department of Biochemistry and Molecular Biology and the Huck Center for Malaria Research at Penn State (The Pennsylvania State University), which allowed the use of their inhouse MS system and database for targeted metabolite annotation. First, an exploration of plant extracts and fractions were studied in parallel to the known active antiplasmodial compounds present in those plants to assess how early in plant screening a potential mode of action can be hypothesized. Results show that extract effects can already be distinguished from a control, though fractions with active compounds separate more clearly. The more purified the fraction, the bigger the correlation in mode of action with the isolated natural compounds, possibly due to the lack of matrix effects and interactions, which shows that metabolomics can be introduced early in bioassay-guided fractionation in plant studies. Second, two of the most important plants in traditional medicine against malaria were studied, Artemisia afra and A. annua, to investigate their phenolic content, presence or absence of artemisinin and correlate the composition with the metabolomic results on a synchronized malaria culture. Results indicate a correlation between activity and artemisinin abundance in these extracts, with A. annua presenting a similar parasitic profile to artemisinin whereas A. afra, despite trace amounts of this compound, differs significantly. A. afra affects various unspecific metabolites but significantly changes myo-inositol distinctly from A. annua and artemisinin, which clearly target redox mechanisms. Lastly, three independent studies aimed to investigate three natural scaffolds for their mode of action: alkyl cyclohexenones compounds named poupartones, ellagic acid and derivatives, and a mixture of triterpene esters. Poupartones showed to interfere with hemoglobin metabolism, DNA and RNA synthesis and redox management systems which correlated to their potential to participate in nucleophilic additions that establish covalent bonds with proteins and generate radical oxygen species, an effective yet not specific type of activity. Our studies on ellagic acid and derivatives support literature data and point to the parasite’s digestive vacuole as the site of action of these compounds. Changes in hemoglobin metabolism and redox metabolites suggest possible effects on plasmepsins, enzymes that act early in hemoglobin breakdown, and on glutathione metabolism, essential to maintaining a balanced organelle. Lastly, a mixture of 8 triterpenic esters seems to affect pyrimidine synthesis and amino acid metabolism through N-carbamoyl-L-aspartate, though it is unclear exactly how. Metabolomics is a hypothesis generating approach that gives a snapshot of the effects of innovative natural compounds on the malaria parasite in order to accurately guide antimalarial drug discovery.
