Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box

Abstract

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...

H3K36 Methylation Reprograms Gene Expression to Drive Early Gametocyte Development in Plasmodium Falciparum

“…Here, we con rmed the dynamic yet stage-speci c nature of H3K36me2&3 (11) and delineate their roles in the transcriptional reprogramming occurring post-commitment in the gametocyte sexual stage of P. falciparum blood-stage development (34). Our chemical interrogation of P. falciparum HDMs supports these conclusions, highlights the importance of the dynamics of histone methylation for transcriptional control and links aberrant H3K36me2&3 patterns with the increased potency of Jumonji HDM inhibitors towards gametocytes reported before (38,65,66).…”

“…JIB-04 (pan-selective) and ML324 (targeting KDM4, PfJMJ3) resulted in the hypermethylation of both H3K36me2&3 relative to the untreated controls (36-to 89-fold, Fig. 5b, Figure S6A), similar to hypermethylation of H3K4me3 and H3K9me3 induced by JIB-04 and ML324 in asexual parasites and gametocytes, respectively (38,66). This hypermethylation was not observed for GSK-J4 (KDM6, H3K27me3 selective, (67) or PCPA-2 (targeting LSD1).…”

“…To investigate the functional relevance of these enzymes for H3K36me2&3, we chemically interrogated HDM activity and determined the effects of this inhibition on H3K36 methylation levels on days associated with stage II gametocyte development. Jumonji HDM inhibitors with activity against gametocytes (38,65,66). JIB-04 (pan-selective) and ML324 (targeting KDM4, PfJMJ3) resulted in the hypermethylation of both H3K36me2&3 relative to the untreated controls (36-to 89-fold, Fig.…”

“…In the asexual parasites, the inhibition of PfJMJ3 by JIB-04 results in transcriptional de-regulation (38). Since this inhibitor is signi cantly more potent towards the sexual stages (38,66), we were particularly interested in the transcriptional effects of JIB-04 in gametocytes since this inhibitor is signi cantly more active against the sexual stages. As such, we performed genome-wide transcriptional pro ling of JIB-04 activity on day 3 and 4 gametocytes each following 24 h treatment with JIB-04 (Additional le 3: Table S6).…”

Natural Products: A Potential Source of Malaria Transmission Blocking Drugs?

“…Alternative sources are the de novo screen of natural product libraries in medium-to high-throughput format. It is now clearly indicated that driving screens based on asexual stage potency may not identify transmission-blocking specific compounds and as such we recommend parallel screens against different parasite life cycle stages or at the start, screening driven primarily on the transmissible forms, after which activity against other stages can be determined for hits obtained [131,132]. For such natural product libraries, stringent go/no go criteria need to be defined, similar to screening any other small molecule libraries.…”