Clinical studies and mathematical models predict that, to achieve malaria elimination, combination therapies will need to incorporate drugs that block the transmission of Plasmodium falciparum sexual stage parasites to mosquito vectors. Efforts to measure the activity of existing antimalarials on intraerythrocytic sexual stage gametocytes and identify transmission-blocking agents have, until now, been hindered by a lack of quantitative assays. Here, we report an experimental system using P. falciparum lines that stably express gametocyte-specific GFP-luciferase reporters, which enable the assessment of dose-and time-dependent drug action on gametocyte maturation and transmission. These studies reveal activity of the first-line antimalarial dihydroartemisinin and the partner drugs lumefantrine and pyronaridine against early gametocyte stages, along with moderate inhibition of mature gametocyte transmission to Anopheles mosquitoes. The other partner agents monodesethyl-amodiaquine and piperaquine showed activity only against immature gametocytes. Our data also identify methylene blue as a potent inhibitor of gametocyte development across all stages. This thiazine dye almost fully abolishes P. falciparum transmission to mosquitoes at concentrations readily achievable in humans, highlighting the potential of this chemical class to reduce the spread of malaria.artemisinin-based combination therapies | transfection

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target–inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.

Summary A comprehensive map of transcription start sites (TSS) across the highly AT-rich genome of P. falciparum would aid progress towards deciphering the molecular mechanisms that underlie the timely regulation of gene expression in this malaria parasite. Using high-throughput sequencing technologies, we generated a comprehensive atlas of transcription initiation events at single nucleotide-resolution during the parasite intra-erythrocytic developmental cycle. This detailed analysis of TSS usage enabled us to define architectural features of plasmodial promoters. We demonstrate that TSS selection and strength are constrained by local nucleotide composition. Furthermore, we provide evidence for coordinate and stage-specific TSS usage from distinct sites within the same transcription unit, thereby producing transcript isoforms, a subset of which are developmentally regulated. This work offers a framework for further investigations into the interactions between genomic sequences and regulatory factors governing the complex transcriptional program of this major human pathogen.

The design of new antimalarial combinations to treat Plasmodium falciparum infections requires drugs that, in addition to resolving disease symptoms caused by asexual blood stage parasites, can also interrupt transmission to the mosquito vector. Gametocytes, which are essential for transmission, develop as sexual blood stage parasites in the human host over 8 to 12 days and are the most accessible developmental stage for transmission-blocking drugs. Considerable effort is currently being devoted to identifying compounds active against mature gametocytes. However, investigations on the drug sensitivity of developing gametocytes, as well as screening methods for identifying inhibitors of early gametocytogenesis, remain scarce. We have developed a luciferase-based high-throughput screening (HTS) assay using tightly synchronous stage I to III gametocytes from a recombinant P. falciparum line expressing green fluorescent protein (GFP)-luciferase. The assay has been used to evaluate the earlystage gametocytocidal activity of the MMV Malaria Box, a collection of 400 compounds with known antimalarial (asexual blood stage) activity. Screening this collection against early-stage (I to III) gametocytes yielded 64 gametocytocidal compounds with 50% inhibitory concentrations (IC 50 s) below 2.5 M. This assay is reproducible and suitable for the screening of large compound libraries, with an average percent coefficient of variance (%CV) of <5%, an average signal-to-noise ratio (S:N) of >30, and a Z= of ϳ0.8. Our findings highlight the need for screening efforts directed specifically against early gametocytogenesis and indicate the importance of experimental verification of early-stage gametocytocidal activity in the development of new antimalarial candidates for combination therapy. P lasmodium falciparum malaria remains a primary global cause of death and disability from infectious disease, particularly in infants and pregnant women (1). Malaria treatment currently relies on artemisinin-based combination therapy (ACT); however, emerging resistance to artemisinins in the field (2, 3) underscores the need to progress new antimalarial candidates through the drug development pipeline. Recent in vitro high-throughput screening (HTS) campaigns against P. falciparum asexual blood stages, the forms responsible for the clinical manifestations of the disease, have identified a wealth of active chemical classes, representing a promising starting point for the discovery of new therapeutic agents (4-7). The current malaria elimination strategy has also highlighted the need for all new antimalarial combination therapies to include components capable of interrupting the transmission of sexual-stage gametocytes to Anopheles mosquitoes (8, 9). Methods to enable prioritization of inhibitors of the asexual parasite stages based on their additional transmission-blocking activity are therefore urgently required.P. falciparum gametocytes develop through five morphologically distinct stages in the human blood over 8 to 12 days (10) and thereafter ...

Equilibrative transporters are potential drug targets, however most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64,560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2–2 µM). These nine compounds completely blocked [3H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5–50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5–50 µM). Wild-type (WT) parasite IC50 values were up to four-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.