Pharmacometabolomic study of the human malaria parasite, Plasmodium falciparum: new insights into parasite biology and mode of drug action

Malaria is a vector-borne parasitic disease spread by a bite of an infected female Anopheles mosquito that accounts for high morbidity and mortality, mainly in sub-Saharan Africa. Of the five species that can cause malaria in humans, Plasmodium falciparum is regarded the most virulent species. Antimalarial drugs, unaltered for many decades, remain the mainstay for treating P. falciparum infection. In addition, despite intensive research there remain significant knowledge gaps in understanding of the biology of the malaria parasite P. falciparum. These deficiencies hinder the ability of scientists to identify new targets for drug discovery at a time when new targets are urgently required, such as in the case of newly emerging drug resistant parasite strains. Therefore, an understanding of the biology of P. falciparum is helpful in identifying new drug targets. Metabolomics, defined as the comprehensive analysis of all metabolites in a biological system, offers a feasible platform for highly sensitive and specific analysis of the metabolic pathways of P. falciparum. This is supported by the assumption that metabolites are important players in biological systems and that resistant parasite strains may operate or alter single or multiple metabolic pathways in order to adapt to the drugs being used. Therefore, a targeted metabolomics approach was developed and validated (Chapter 3) in order to better understand the metabolic roles of mitochondria and the digestive vacuole of P. falciparum. Metabolite detection and quantification were conducted using a targeted LC-MS/MS metabolomics approach (Chapter 3). It was shown that metabolic activities, particularly carbohydrate metabolism, in trophozoite stage P. falciparum-infected RBC were remarkably higher than that of non-infected RBC (Chapter 4). This lead the study to progress further, examining the metabolic role of two components of the P. falciparum; the mitochondria and the digestive vacuole. A number of mitochondrial inhibitors selective for specific electron transport chain complexes and mitochondrial transporters were used to assess mitochondrial function in asexually growing trophozoite stage P. falciparum (Chapter 5 and 6). Despite the differing modes of action of the inhibitors, the metabolic fingerprints, which were carbamoyl-l-aspartate and dihydroorotate, from these experiments were consistent with the parasite mitochondrion playing a key role in pyrimidine biosynthesis at the point of dihydroorotate dehydrogenase (DHODH) (Chapter 5 and 6). This metabolic fingerprint, leading to parasite death, was quite distinct from fingerprints obtained from biologically distinct inhibitors such as heme-binding drugs (quinoline-containing antimalarials drugs) which primarily affected the metabolism of amino acids, perhaps in the digestive vacuole and parasite cytosol (Chapter 7). In contrast to genomic and proteomic approaches, metabolomics appears to better represent the parasites’ phenotype in response to drug perturbation. Pharmacometabolomics will therefore have significant utility in understanding the biological function of parasite components; and the mode of action, efficacy and toxicity of pharmaceutical drugs