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Abstract

Despite global efforts for its elimination, malaria continues to be a major infectious disease worldwide. In 2020, more than 240 million cases and more than 600 000 deaths were reported. This represents a setback in the progress observed in the past decades, which had also reached a plateau in the past few years. This data also shows that the milestones set in the World Health Organization’s Global Technical Strategy for Malaria 2016-2030, a plan adopted by the United Nations, have not been met. With an increase in mosquito resistance to insecticides and parasite resistance to antimalarial therapies as main biological threats to malaria control, there is a dire need of new medicines with novel modes of action that are refractory to resistance development. SC83288 is a promising clinical candidate for the treatment of severe malaria that has a novel structure. With high in vitro activity against multiple P. falciparum strains and no evidence of cross-resistance with other known antiplasmodial drugs, the compound appears to have a novel mode of action as well. The main objective of this doctoral thesis was to gain a better understanding of the antimalarial mechanism of SC83288 and to identify its molecular targets. To this end, a wide range of methodologies were employed. The analysis of the potential relationship of the compound with antifolates led to the observation that it has an affinity for ATP-binding sites, such as those present in kinases. An untargeted metabolomics study revealed that upon treatment with SC83288, parasites accumulated products of the metabolism of AdoMet through a pathway that has not been reported before in P. falciparum. In addition, treated parasites presented lower levels of phosphatidylserine lipids and intermediates of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Live cell imaging showed that early-treated parasites were not able to undergo DNA replication and displayed membrane disruptions accompanied by cell death. A label-free proteomic approach revealed that SC83288 interacts with a number of parasite proteins, from which four were identified as potential targets: fumarate hydratase, protein kinase CK2, DNA replication licensing factor MCM2 and cytosolic [Fe-S] protein assembly protein CIA1. An analysis of the transcriptional response of P. falciparum to SC83288 revealed a major downregulation of transcription, and a correlation to the gene transcription profile elicited by methylene blue. Taken together, these results led to the formulation of the hypothesis that the main target of SC83288 is the plasmodial enzyme fumarate hydratase. It is additionally proposed that the compound has multiple targets, which may include CK2, MCM2 and CIA1. Although further experimental evidence is needed to validate this hypothesis, the results represent a substantial leap towards the elucidation of the mode of action of SC83288.