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To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum , is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817 -resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817 , and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial., Competing Interests: The authors declare no conflict of interest.

Current best practice for the treatment of malaria relies on short half-life artemisinins that are failing against emerging Kelch 13 mutant parasite strains. Here, we introduce a liposome-like self-assembly of a dimeric artesunate glycerophosphocholine conjugate (dAPC-S) as an amphiphilic prodrug for the short-lived antimalarial drug, dihydroartemisinin (DHA), with enhanced killing of Kelch 13 mutant artemisinin-resistant parasites. Cryo-electron microscopy (cryoEM) images and the dynamic light scattering (DLS) technique show that dAPC-S typically exhibits a multilamellar liposomal structure with a size distribution similar to that of the liposomes generated using thin-film dispersion (dAPC-L). Liquid chromatography-mass spectrometry (LCMS) was used to monitor the release of DHA. Sustainable release of DHA from dAPC-S and dAPC-L assemblies increased the effective dose and thus efficacy against Kelch 13 mutant artemisinin-resistant parasites in an in vitro assay. To better understand the enhanced killing effect, we investigated processes for deactivation of both the assemblies and DHA, including the roles of serum components and trace levels of iron. Analysis of parasite proteostasis pathways revealed that dAPC assemblies exert their activity via the same mechanism as DHA. We conclude that this easily prepared multilamellar liposome-like dAPC-S with long-acting efficacy shows potential for the treatment of severe and artemisinin-resistant malaria.

The peroxide bond of the artemisinins inspired the development of a class of fully synthetic 1,2,4-trioxolane-based antimalarials, collectively known as the ozonides. Similar to the artemisinins, heme-mediated degradation of the ozonides generates highly reactive radical species that are thought to mediate parasite killing by damaging critical parasite biomolecules. We examined the relationship between parasite dependent degradation and antimalarial activity for two ozonides, OZ277 (arterolane) and OZ439 (artefenomel), using a combination of in vitro drug stability and pulsed-exposure activity assays. Our results showed that drug degradation is parasite stage dependent and positively correlates with parasite load. Increasing trophozoite-stage parasitemia leads to substantially higher rates of degradation for both OZ277 and OZ439, and this is associated with a reduction in in vitro antimalarial activity. Under conditions of very high parasitemia (∼90%), OZ277 and OZ439 were rapidly degraded and completely devoid of activity in trophozoite-stage parasite cultures exposed to a 3-h drug pulse. This study highlights the impact of increasing parasite load on ozonide stability and in vitro antimalarial activity and should be considered when investigating the antimalarial mode of action of the ozonide antimalarials under conditions of high parasitemia., (Copyright © 2018 American Society for Microbiology.)

Fully synthetic endoperoxide antimalarials, namely, OZ277 (RBx11160; also known as arterolane) and OZ439 (artefenomel), have been approved for marketing or are currently in clinical development. We undertook an analysis of the kinetics of the in vitro responses of Plasmodium falciparum to the new ozonide antimalarials. For these studies we used a K13 mutant (artemisinin resistant) isolate from a region in Cambodia and a genetically matched (artemisinin sensitive) K13 revertant. We used a pulsed-exposure assay format to interrogate the time dependence of the response. Because the ozonides have physicochemical properties different from those of the artemisinins, assay optimization was required to ensure that the drugs were completely removed following the pulsed exposure. Like that of artemisinins, ozonide activity requires active hemoglobin degradation. Short pulses of the ozonides were less effective than short pulses of dihydroartemisinin; however, when early-ring-stage parasites were exposed to drugs for periods relevant to their in vivo exposure, the ozonide antimalarials were markedly more effective., (Copyright © 2016 Yang et al.)