Summary The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Highlights d Mutations in PfAcAS confer resistance to antiplasmodials MMV019721 and MMV084978 d MMV019721 and MMV084978 specifically inhibit PfAcAS by competing with substrates d cKD and IFA show PfAcAS is an essential nuclear enzyme in blood-stage parasites d PfAcAS inhibitors deplete parasite acetyl-CoA and result in histone hypoacetylation

39New chemical matter is needed to target the divergent biology associated with the different 40 life cycle stages of Plasmodium. Here, we report the parallel screening of the Medicines for 41 Malaria Venture Pandemic Response Box to identify multistage-active and stage-specific 42 compounds against various life cycle stages of Plasmodium parasites (asexual parasites, 43 stage IV/V gametocytes, gametes, oocysts and liver stages) and for endectocidal activity. Hits 44 displayed unique chemotypes and included two multistage-active compounds, 16 asexual-45 targeted, six with prophylactic potential and ten gametocyte-targeted compounds. Notably, 46 four structurally diverse gametocyte-targeted compounds with potent transmission-blocking 47 activity were identified: the JmjC inhibitor ML324, two azole antifungals including 48 eberconazole, and the antitubercular clinical candidate SQ109. Besides ML324, none of these 49 have previously attributed antiplasmodial activity, emphasizing the success of de novo parallel 50 screening against different Plasmodium stages to deliver leads with novel modes-of-action. 51Importantly, the discovery of such transmission-blocking targeted compounds covers a 52 previously unexplored base for delivery of compounds required for malaria elimination 53 strategies. 55 56Malaria treatment solely relies on drugs that target the parasite but current treatment options 57 have a finite lifespan due to resistance development. Moreover, whilst current antimalarials 58 are curative of asexual blood stage parasitemia and associated malaria symptoms, they 59 cannot all be used prophylactically and typically do not effectively block transmission. This 60 limits their utility in malaria elimination strategies, where the latter dictates that chemotypes 61 should block human-to-mosquito (gametocyte and gametes) and mosquito-to-human 62 (sporozoites and liver schizonts) transmission. 63The transmission stages of malaria parasites are seen as parasite population 64 bottlenecks, 1 with as few as 100 sporozoites able to initiate an infection after migrating to the 65 liver where exoerythrocytic schizogony occurs. The subsequent release of thousands of 66 daughter cells, which in turn infect erythrocytes, initiates the extensive population expansion 67 that occurs during asexual replication. A minor proportion (~1%) 2 of the proliferating asexual 68 parasites will undergo sexual differentiation to form mature stage V gametocytes, a 10-14 day 69 process in the most virulent parasite Plasmodium falciparum. Only ~10 3 of these falciform-70 shaped mature gametocytes are taken up by the next feeding mosquito to transform into male 71 and female gametes in the mosquito's midgut. 3 Fertilization results in zygote development, 72 and a motile ookinete that passes through the midgut wall forms an oocyst from which 73 sporozoites develop, making the mosquito infectious.74 The sporozoite and gametocyte population bottlenecks have been the basis of enticing 75 arguments towards the development of chemotypes able to targe...

In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage P. falciparum parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance emerged. Whole genome sequencing revealed all 17 resistant clones acquired nonsynonymous mutations in the gene encoding the orphan apicomplexan transporter PF3D7_0312500 ( pfmfr3 ) predicted to encode a member of the major facilitator superfamily (MFS). Disruption of pfmfr3 and testing against a panel of antimalarial compounds showed decreased sensitivity to MMV085203 and GNF-Pf-3600 as well as other compounds that have a mitochondrial mechanism of action. In contrast, mutations in pfmfr3 provided no protection against compounds that act in the food vacuole or the cytosol. A dihydroorotate dehydrogenase rescue assay using transgenic parasite lines, however, indicated a different mechanism of action for both MMV085203 and GNF-Pf-3600 than the direct inhibition of cytochrome bc1. Green fluorescent protein (GFP) tagging of PfMFR3 revealed that it localizes to the parasite mitochondrion. Our data are consistent with PfMFR3 playing roles in mitochondrial transport as well as drug resistance for clinically relevant antimalarials that target the mitochondria. Furthermore, given that pfmfr3 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.