Malaria is a major global health burden, responsible for over 200 million cases annually, and new antimalarial medicines are urgently needed to treat this potentially-fatal parasitic infection. The malaria parasite, Plasmodium falciparum, has developed resistance to all currently-approved antimalarials, which underscores the critical need to discover new drugs with novel mechanisms of action. The bis-triazines represent a novel class of antimalarial compounds that were identified in a high-throughput phenotypic screen and further optimised to exhibit potent antimalarial activity. Importantly, these compounds are based on a novel chemical scaffold, but the mechanism of action of is unknown, which limits further development of this series. The aim of this study was to reveal the mechanism of action of bis-triazine antimalarials.
An untargeted metabolomics approach using high resolution accurate mass LC-MS was implemented to reveal the impact of bis-triazine treatment on the cellular biochemistry of P. falciparum. Incubation of P. falciparum-infected red blood cells with a potent bis-triazine induced a unique metabolic profile that differed from other known antimalarials. A dose-dependent accumulation of dimethyl-arginine was the most significant unique metabolic perturbation observed in treated cells. Levels of related metabolites, including monomethylated arginine and lysine, were also increased following compound exposure. Stable-isotope tracing and proteomic studies confirmed the perturbation of methylation pathways, and fluorescent labelling of bis-triazine analogues using click chemistry revealed localisation in the parasite nucleus. Overall, this work reveals a novel mechanism of action for the bis-triazine antimalarials, leading to aberrant protein methylation within the parasite.