The increasing prevalence of resistance to antimalarial medicines underscores a critical need to discover new drugs with novel mechanisms of action. We have identified a novel series of bis-triazines with potent antimalarial activity in vitro (Plasmodium falciparum IC50: <10 nM) and in vivo (P. berghei Peters 4-day test, mouse, ED50: 0.21 mg/kg/day by sc route and 1.47 mg/kg/day by po route). The aim of this work was to identify the mechanism of action of these novel compounds.
As these bis-triazines are structurally distinct from other known antimalarials, and no prior mechanistic information was available, the mode of action was explored using a combination of untargeted metabolomics, proteomics, chemical proteomics and fluorescence microscopy.
Incubation of P. falciparum-infected red blood cells with a potent bis-triazine compound induced a unique metabolomic 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, and stable-isotope tracing suggested inhibition of demethylase activity. Gene ontology enrichment analysis of the proteomics data revealed downregulation of several nucleic-acid binding proteins in treated parasites compared to controls. Bifunctional bis-triazine analogues bearing photoreactive and ‘click chemistry’ motifs were synthesised and demonstrated excellent activity against P. falciparum in vitro (IC50: 12 nM). These probes allowed photo-activated immobilisation of the active compound within cells, and visualisation with ‘click chemistry’-linked fluorescent dyes, which revealed co-localisation with the parasite nucleus. Further work is ongoing to isolate and identify the specific nuclear protein targeted by these compounds.
Overall, this work reveals a novel mechanism of action for the bis-triazine antimalarials, demonstrating that these compounds target the parasite nucleus and cause aberrant arginine methylation. This mechanism differs from other known antimalarials and supports further optimisation of this attractive novel compound series.