As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapsein a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

Gene expression in Plasmodia integrates post-transcriptional regulation with epigenetic marking of active genomic regions through histone post-translational modifications (PTMs). To generate insights into the importance of histone PTMs to the entire asexual and sexual developmental cycles of the parasite, we used complementary and comparative quantitative chromatin proteomics to identify and functionally characterise histone PTMs in 8 distinct life cycle stages of P. falciparum parasites. ~500 individual histone PTMs were identified of which 106 could be stringently validated. 46 individual histone PTMs and 30 co-existing PTMs were fully quantified with high confidence. Importantly, 15 of these histone PTMs are novel for Plasmodia (e.g. H3K122ac, H3K27me3, H3K56me3). The comparative nature of the data revealed a highly dynamic histone PTM landscape during life cycle development, with a set of histone PTMs (H3K4ac, H3K9me1 and H3K36me2) displaying a unique and conserved abundance profile exclusively during gametocytogenesis (P < 0.001). Euchromatic histone PTMs are abundant during schizogony and late gametocytes; heterochromatic PTMs mark early gametocytes. Collectively, this data provides the most accurate, complete and comparative chromatin proteomic analyses of the entire life cycle development of malaria parasites. A substantial association between histone PTMs and stage-specific transition provides insights into the intricacies characterising Plasmodial developmental biology.

BackgroundThe discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions.MethodsGametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable.ResultsA highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4 %. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident.ConclusionsWith this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broad-diversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0718-z) contains supplementary material, which is available to authorized users.

The emergence of resistance toward artemisinin combination therapies (ACTs) by the malaria parasite has the potential to severely compromise malaria control. Therefore, the development of new artemisinins in combination with new drugs that impart activities toward both intraerythrocytic proliferative asexual and transmissible gametocyte stages, in particular, those of resistant parasites, is urgently required. We define artemisinins as oxidant drugs through their ability to oxidize reduced flavin cofactors of flavin disulfide reductases critical for maintaining redox homeostasis in the malaria parasite. Here we compare the activities of 10-amino artemisinin derivatives toward the asexual and gametocyte stages of parasites. Of these, artemisone and artemiside inhibited asexual and gametocyte stages, particularly stage V gametocytes, in the low-nanomolar range. Further, treatment of both early and late gametocyte stages with artemisone or artemiside combined with the pro-oxidant redox partner methylene blue displayed notable synergism. These data suggest that modulation of redox homeostasis is likely an important druggable process, particularly in gametocytes, and this finding thereby enhances the prospect of using combinations of oxidant and redox drugs for malaria control.

Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis, and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine synthetase. Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.Polyamines such as putrescine, spermidine, and spermine are small organic compounds containing two or more amino groups. At physiological pH, these polycations interact electrostatically with numerous anionic macromolecules, thereby stabilizing DNA, RNA, nucleoside triphosphates (e.g. ATP), phospholipids, and proteins (1, 2). These interactions with polyamines can alter DNA conformation, regulate replication and transcription, strengthen membranes, regulate ion channels, and protect DNA and phospholipids from oxidative stress (1-5). Yet polyamines are also implicated in apoptosis (5). Polyamine depletion generally causes cytostasis or growth arrest, which implies that these molecules are involved in cell cycle progression and regulation, and it is speculated that polyamines regulate cyclin degradation (1, 6, 7). Therefore, polyamines are essential for cellular growth, differentiation, and macromolecular synthesis and are ubiquitous components of all living cells, except two orders of Archaea (1). Polyamine metabolism is particularly important in rapidly proliferating cells and has been exploited in the treatment of cancer (1) and parasitic diseases (8). Polyamine metabolism of the malaria parasite Plasmodium falciparum is also a potential target for therapeutic intervention (9, 10).Polyamine and methionine metabolism are closely connected. This is particularly evident in Plasmodium where the two rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) 2 and S-adenosylme...