Malaria 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.
The 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 compounds. These efforts rely on selective and predictive assays for gametocytocidal 93 activity, 4 transmission-blocking, 5, 6 and hepatic development. 7 Moreover, identifying stage-94 specific compounds will allow the divergent cell biology associated with the different life cycle 95 states to be targeted. 5, 6, 8 Recently, parallel screening of diversity sets has resulted in reports 96 of such stage-specific compounds. 7, 9, 10, 11 122 screened all compounds on three independent gametocyte assay platforms to confirm that hit 123 selection (compounds active on at least 2 platforms) was independent of assay readout. 4 124 Primary hits were identified with a relatively lenient but inclusive cut-off of ³50% inhibition (at 125 2 μM for asexual stages and 5 μM for gametocytes and liver stages, the latter at >60% cut-126 off). Asexual stage activity was confirmed against drug resistant PfDd2 asexual parasites. potential of compounds with gametocytocidal activity was confirmed by inhibition of male 129 gamete exflagellation and in a standard membrane feeding assay (SMFA).
An 18% hit rate was obtained against PfNF54 asexual parasites, 12% against PfNF54 131 stage IV/V gametocytes and 11% against liver stages (Figure 2A , Supplementary file S1).
Although a number of compounds showed activity against all these life cycle stages, stage- 
Based on the target indicators / biological pathway descriptors available for the 144 compounds in the PRB in other diseases, PfNF54 asexual hits were enriched for inhibitors of 145 kinases, CYP450, energy metabolism and DNA synthesis. Inhibitors of dihydrofolate 146 reductase (DHFR, antifolates), dihydroorotate dehydrogenase (DHODH), proton pumps and 147 topoisomerase were exclusively hits for PfNF54 asexual parasites and liver stages.
Compounds with both PfNF54 asexual and gametocyte activity include antithrombotics, 149 protease inhibitors, sphingosine-1-phosphate receptor modulators and compounds affecting 150 redox homeostasis, whereas inhibitors of MmpL3 (mycobacterial membrane protein large 3) 151 and ion channels were predominant in gametocyte hits ( Figure 2C ). Interestingly, well known 152 antimicrobials (e.g. thalidomide, isoniazid and saquinavir) were not active in our screens.
Chemical classes highly represented in the hit pool include quinolines, benzamides/benzoids 154 and azoles. propargyl-linked antifolate, 15 MMV1580844 (IC50 = 0.0017 μM), which targets DHFR in 176 mammalian and yeast cells. 16, 17 It also showed activity against P. berghei liver stages (0.004 177 μM). As there was no activity against PfNF54 stage IV/V gametocytes, our data concur that 178 inhibition of Plasmodium DHFR is only important to asexual and liver stage schizogony. 6 179 MMV1580844 had a pronounced (63-fold) loss of activity against the antifolate 180 (pyrimethamine) resistant PfDd2 line. By contrast, the quinazoline antifolate trimetrexate 181 (MMV1580173, derived from methotrexate) was potently active against PfDd2 (IC50 = 0.108 182 μM) as well as P. berghei liver stages (IC50 = 0.0005 μM), in both instances with more than 183 10-fold selectivity towards the parasite versus CHO cells. As with MMV1580844,
MMV1580173 did not display any gametocytocidal activity.
The majority (13/16) of the asexual-specific compounds have not been previously 
To determine if the compounds also directly targeted gametes, the assay was repeated 
The compounds with transmission-blocking potential were additionally evaluated for their 270 activity as endectocides, killing mosquitoes after being supplied in a blood meal. However, 271 none of the compounds produced significant mortality (one-way ANOVA, p = 0.7005, total 272 DF=71, n³2) in the 4-day mortality assay at 2 μM compared to DMSO as control under these MmpL3 inhibitors MMV687273 (SQ109) and MMV1580843, the GPCR inhibitor 275 MMV1581558, AZD-0156 (MMV1580483) and MMV1174026, marking these compounds with 276 a potential to kill the parasite as well as the mosquito vector. Interestingly, all these compounds 277 also had >60% reduction of TRA in addition to their potential endectocidal activity.
The ability to quickly respond to pandemics has become of paramount importance, and 281 compound sets like the MMV PRB provide an essential tool to support rapid screening of 282 diverse druggable compounds for potential repurposing. Indeed, antimalarials have previously 
Multistage activity is a preferential attribute for the next generation of antimalarials, 36 290 but such compounds are rarely found in diversity library screens, in large part due to targeted 291 screening approaches rather than parallel screening in multiple assays. We identified two non- Plasmodium selectivity in an analogue program directed against malaria will be challenging.
In addition to selectivity over human homologues, MMV1593539 also contains a nitro group 328 that is considered a red flag, and safety needs to be assessed or attempts made to replace 329 this group. Interestingly, of the two reported pyruvate kinases in P. falciparum (PK1 and PK2), 330 PK1 has a crystal structure (10.2210/pdb3KHD/pdb), which could guide selectivity and 331 optimization studies provided PfPK1 is responsible for phenotypic activity. 
Our stringent profiling cascade additionally ensured a high success rate in confirming 342 transmission-blocking activity and validates the use of orthogonal gametocytocidal screens 4, 343 11 as a primary filter in large scale screens. In addition, this approach resulted in a linear 344 correlation between gametocytocidal activity and activity against male gametes, which directly 345 translated to oocyst reduction. By evaluating both TRA and TBA, our data highlighted the carried by such mosquitoes will result in a lowered efficacy of transmission, as intensity of a 349 mosquito infection has been shown to be critically important to the success of transmission. 40 350 However, we additionally observe a decrease in oocyst prevalence (TBA), that implies that 351 the majority of mosquitoes treated with compounds that affect oocyst prevalence would not 352 carry parasites. The latter will also therefore have an epidemiological impact in line with WHO- 
Our data also indicate specific gametocyte-associated biological processes worthy of 371 further investigation. Though the PRB contained nine azole antifungals (including miconazole, 372 ketoconazole, fluconazole), only two imidazoles (eberconazole, MMV1634492, and 373 MMV1634491) showed activity against gametocytes, the latter with >10-fold selectivity relative 374 to the asexual stage. This is the first report of the antimalarial activity of eberconazole, though 375 this has been seen for other azole antifungals. Ketoconazole, miconazole and clotrimazole, 376 like eberconazole, inhibit fungal ergosterol biosynthesis, 24 but they are also known to target 377 the heme detoxification system in asexual P. falciparum. 43 Although heme catabolism is 378 essentially absent in mature gametocytes, 5 some heme metabolic activity seems to be present 379 in P. berghei mosquito stages 44 and this process as a target for these compounds cannot be 380 excluded. Moreover, as these compounds have exclusive transmission-blocking activity, a 381 novel mode of action might be indicated.
In addition to the azoles, we identified ML324 with exclusive activity against that ML324 has increased potency as gametocytes mature to stage IV/V (0.077 μM) and 386 potently kills male gametes with confirmed transmission-blocking activity. This implies that 387 mature gametocytes and male gametes are even more sensitive to changes in histone 388 methylation status due to ML324 treatment, which results in deregulated gene expression, 
(Malaria Drug Accelerator) will be critical to progress these compounds from discovery to 412 development. One advantage of screening boxes such as the PRB that contain a large number 413 of compounds with already well described DMPK profiles and empirically determined physical 414 characteristics, is that they could progress rapidly through the drug discovery pipeline. 
Chloroquine and artesunate were used as controls.
PfDd2 asexual parasites activity was determined with SYBR Green I as described 13 
Potential causal prophylactic activity was tested as previously described 13 on HepG2-A16-484 CD81 cells exposed to compounds and incubated for 24 h. Thereafter, fresh P. 
Emetine was used as the control. Cytotoxicity against a Hepatocellular carcinoma line 496 (HepG2-A16-CD81) was performed by adding Promega CellTiter-Glo ® (2 μL) to these cells 497 seeded as above for the sporozoite liver stage assay but in the absence of sporozoites, and 498 luminescence measured. Curve fitting was done as above using puromycin (positive control) 499 and DMSO (negative control). 
All computer codes used to analyze the data are available from the corresponding author upon 543 request 544 Data availability
The data presented in this study area available from the corresponding author upon request. 