With the emergence of widespread resistance to all current antimalarials, there is increased urgency to develop new drugs with novel mechanisms of action. Second generation analogues of the bis-1,2,4-triazines are a new class of antimalarials with fast-killing activity and nanomolar potency against the deadliest malaria parasite species, Plasmodium falciparum. However, the novel mechanism of action (MOA) of our series remains unknown and some molecules exhibit parasite-mediated instability. To address these challenges and understand the interplay between antimalarial activity and intraparasitic stability, we are employing LC-MS-based metabolomics and proteomics methods.
Using a targeted LC-MS method, we monitored compound loss over time in infected red blood cell (iRBC) cultures with increasing levels of parasitaemia. For selected compounds, instability was amplified with a ten-fold increase in parasite load which in turn, decreased antimalarial activity by ten-fold over a 5-hour drug treatment, indicating a relationship exists between stability and activity. Further investigation into the mechanism of instability will include uncovering the drug-derived metabolites through untargeted metabolomics.
To identify the molecular target, we used solvent protein profiling (SPP) on the latest Orbitrap Astral MS technology. This method was validated using the known antimalarial cytochrome bc1 inhibitor, atovaquone, which selectively stabilised 8 proteins of the parasite cytochrome bc1 complex from 4624 proteins reproducibly detected from a parasite proteome lysate, representing near complete coverage of the iRBC proteome. With the triazine analogue, a handful of significantly stabilised proteins were identified as potential targets. Consistent with previous findings for the bis-1,2,4-triazines, nuclear proteins involved in transcription and RNA-processing were identified as hits. Further confirmation of these targets will be carried out through additional proteomics and transcriptomics studies. Understanding the process that underpins instability and its relationship with activity could be crucial to determining the MOA and progressing this series towards clinical evaluation.