Malaria caused approximately 435 000 deaths in 2017 and resistance has now emerged to the current first-line artemisinin-based antimalarials, highlighting the urgent need for new malaria therapeutics. The ozonides are a novel class of synthetic antimalarial drugs with potent activity against the deadliest malaria parasite species, Plasmodium falciparum, but their mechanism of action is poorly defined. In this study, the mode of action of OZ277 (marketed in India) and OZ439 (in Phase IIb clinical trials) were examined in P. falciparum infected red blood cells using an untargeted multi-omics approach consisting of proteomics, peptidomics and time-dependent metabolomics.
Untargeted metabolomic profiling using LC-MS with high resolution accurate mass spectrometry revealed a rapid depletion of short chain haemoglobin-derived peptides and the formation of ozonide-alkylated haem adducts in drug-treated parasite cultures. A dedicated peptidomics method was also developed and revealed drug-induced accumulation of long chain haemoglobin-derived peptides. These findings agree with the proposal that ozonides are activated by free haem in the food vacuole of the parasite and initially perturb haemoglobin catabolism. Extended ozonide exposure disrupted secondary biochemical pathways, including lipid metabolism and nucleotide biosynthesis. Quantitative proteomic analysis confirmed ozonide treatment perturbs these pathways, but also revealed an upregulation of proteins involved in translation and the ubiquitin-proteasome system, suggestive of the parasite engaging a proteostatic stress response to mitigate ozonide-induced damage. Furthermore, untargeted chemical proteomic studies showed that ozonides alkylate multiple proteins within the parasite, which likely contributes to ozonide-induced protein stress.
This unbiased multi-omics approach revealed ozonides initially impact haemoglobin digestion, followed by secondary effects on additional biochemical pathways that are critical for parasite survival. These findings provide new insight into the mode of action of ozonides, and facilitates new opportunities for interventions to enhance their antimalarial efficacy and reduce the potential for developing resistance.