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.

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.

A novel diazaspiro [3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp 3 -rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure−activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by wholegenome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

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...