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The apicoplast is an essential organelle for the viability of apicomplexan parasites Plasmodium falciparum or Toxoplasma gondii , which has been proposed as a suitable drug target for the development of new antiplasmodial drug-candidates. Plasmodione, an antimalarial redox-active lead drug is active at low nM concentrations on several blood stages of Plasmodium such as early rings and gametocytes. Nevertheless, its precise biological targets remain unknown. Here, we described the synthesis and the evaluation of new heteroaromatic analogues of plasmodione, active on asexual blood P. falciparum stages and T. gondii tachyzoites. Using a bioimaging-based analysis, we followed the morphological alterations of T. gondii tachyzoites and revealed a specific loss of the apicoplast upon drug treatment. Lipidomic and fluxomic analyses determined that drug treatment severely impacts apicoplast-hosted FASII activity in T. gondii tachyzoites, further supporting that the apicoplast is a primary target of plasmodione analogues. To follow the drug localization, "clickable" analogues of plasmodione were designed as tools for fluorescence imaging through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Short-time incubation of two probes with P. falciparum trophozoites and T. gondii tachyzoites showed that the clicked products localize within, or in the vicinity of, the apicoplast of both Apicomplexa parasites. In P. falciparum , the fluorescence signal was also associated with the mitochondrion, suggesting that bioactivation and activity of plasmodione and related analogues are potentially associated with these two organelles in malaria parasites.

Understanding the mechanisms of drug action in malarial parasites is crucial for the development of new drugs to combat infection and to counteract drug resistance. Proteomics is a widely used approach to study host-pathogen systems and to identify drug protein targets. Plasmodione is an antiplasmodial early-lead drug exerting potent activities against young asexual and sexual blood stages in vitro with low toxicity to host cells. To elucidate its molecular mechanisms, an affinity-based protein profiling (AfBPP) approach was applied to yeast and P. falciparum proteomes. New (pro-) AfBPP probes based on the 3-benz(o)yl-6-fluoro-menadione scaffold were synthesized. With optimized conditions of both photoaffinity labeling and click reaction steps, the AfBPP protocol was then applied to a yeast proteome, yielding 11 putative drug-protein targets. Among these, we found four proteins associated with oxidoreductase activities, the hypothesized type of targets for plasmodione and its metabolites, and other proteins associated with the mitochondria. In Plasmodium parasites, the MS analysis revealed 44 potential plasmodione targets that need to be validated in further studies. Finally, the localization of a 3-benzyl-6-fluoromenadione AfBPP probe was studied in the subcellular structures of the parasite at the trophozoite stage., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)