Malaria causes 445 000 deaths annually and threatens approximately 40% of the world population. The malaria parasite has developed resistance to most approved antimalarials, and there is 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 cell and animal models. 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 comparative multi-omics analysis and chemical proteomics.
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 (DMA) was the most significant unique metabolic perturbation observed in treated cells, and levels of monomethylated arginine and lysine were also increased. Stable-isotope tracing suggested that the DMA accumulation was due to inhibition of demethylase activity or increased degradation of methylated proteins. Gene ontology enrichment analysis of the proteomics data revealed depletion of several nucleic-acid binding proteins in treated parasites, and peptidomics revealed accumulation of peptides from methylated nuclear proteins.
Chemical proteomics studies using bifunctional bis-triazine analogues bearing photoreactive and ‘click chemistry’ motifs allowed enrichment of triazine-bound proteins, and LC-MS analysis identified 15 candidate proteins as potential drug targets. Five of these candidates are nuclear proteins, which currently represent the most likely candidates based on the impact on nuclear proteins observed in the multi-omics studies. Fluorescence microscopy has confirmed co-localisation of bis-triazine probes with the parasite nucleus and further work is ongoing to confirm the specific protein target(s) responsible for the action of these promising new antimalarial compounds.