Authors
C Giannangelo1; S A Charman1; D J Creek1; 1 Monash University, Australia Discussion
Artemisinin (ART)-based combination therapies are the current first line treatment for uncomplicated malaria caused by Plasmodium falciparum. However, the emergence and spread of ART resistant parasites threatens their ongoing use. Identifying new antimalarial drugs is therefore vital for effective control and to minimise the burden of disease caused by Plasmodium infection. The potent and fully synthetic peroxide-based ozonide (OZ) antimalarials, OZ277 and OZ439, are active against all blood stages of the parasite and could be promising replacements for the ART antimalarials. However, despite being approved for clinical use (OZ277) and in advanced stages of development (OZ439), detailed information on the biochemical mechanisms underlying the activity of these compounds is lacking. We employed an untargeted metabolomics approach to monitor metabolic perturbations induced by the OZs on P. falciparum asexual blood stage cultures. Liquid chromatography-mass spectrometry allowed observation of drug-dependent changes in metabolite levels over time, enabling the identification of early effects on parasite metabolism to be distinguished from a non-specific death phenotype. We showed that treatment of trophozoite-stage cultures with OZ277 and OZ439 resulted in depletion of short chain peptides, most likely derived from haemoglobin, within 1.5h. Further untargeted peptidomic studies confirmed haemoglobin-derived peptides were perturbed after OZ treatment. Together these data show that haemoglobin catabolic processes are the most significant initial metabolic perturbations induced by OZ antimalarial treatment, which is consistent with the hypothesis that these drugs are activated by Fe(II) to produce reactive intermediates in the digestive vacuole. Further investigations aim to characterise the parasite response to OZ treatment during the ring-stage of asexual development in ART-sensitive and -resistant parasite lines. 
