3‑Benzylmenadiones\nand their Heteroaromatic\nAnalogues Target the Apicoplast of Apicomplexa Parasites: Synthesis\nand Bioimaging Studies

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