A high-throughput compound screen identifies multiple druggable targets in Plasmodium falciparum transmission stages

Seefeldt, L.; Gumpp, C.; Carril, O.; Eberhardt, J.; Boltryk, S.; Passecker, A.; Thommen, B. T.; Sifoniou, K.; Scheurer, C.; Fischli, C.; Babai, D. I.; Mahmoud, A. H.; Alexander, L. T.; Renner, S.; Guiguemde, A. W.; Pei, L.; Gruering, C.; Butendeich, H.; Siebert, D.; Straimer, J.; Tobiasson, V.; Baumgarten, S.; Lill, M. A.; Baeschlin, D. K.; Rottmann, M.; Voss, T. S.; Brancucci, N. M. B.

doi:10.64898/2026.09.10.747198

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bioRxiv · 10.64898/2026.09.10.747198

A high-throughput compound screen identifies multiple druggable targets in Plasmodium falciparum transmission stages

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Abstract

Most antimalarials are ineffective against the sexual transmission stages, known as gametocytes, of the malaria parasite Plasmodium falciparum. Their low sensitivity to drugs is attributed to limited compound uptake and a poorly understood form of cellular quiescence. Our current understanding of druggable transmission-blocking processes is therefore limited. Based on genetically engineered parasites that facilitate the mass production of synchronous mature gametocytes, we developed a high throughput drug screening platform that allowed us to test more than 50,000 compounds for gametocytocidal effects in one day. By screening a diversity-oriented library, we identified over 40 molecules that kill mature gametocytes in the low nanomolar range. Using resistance selection coupled to whole genome sequencing and drug-target interaction modelling, we followed up on three chemically tractable compounds that are also highly active against asexual parasites and prevent gametocyte transmission to mosquitoes. We show that the compound ONX-0914, a specific inhibitor of the {beta}5i/LMP7 subunit of human immunoproteasomes, targets the parasite proteasomal {beta}5 subunit. In contrast, the compounds CR-1-31-B and brusatol interfere with translation by targeting eukaryotic initiation factor 4A (eIF4A) and the peptidyl transferase center (PTC) of the 80S ribosome, respectively. Interestingly, parasite resistance to brusatol, a broad-spectrum antitumor drug, is linked to the differential modification of specific rRNA bases near the ribosomal A-site, mediated by altered base specificity of a rRNA methyltransferase. In summary, we successfully combined high-throughput compound screening with drug target deconvolution to reveal the targets and mode-of-action for three potent gametocytocidal molecules and discover the mechanism of resistance to the anti-tumorigenic drug brusatol. In addition to critically advancing our understanding of mature gametocyte biology and druggable processes in P. falciparum transmission stages, our observations made with brusatol-resistant parasites may become relevant for anti-cancer drug research.

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Seefeldt, L., Gumpp, C., Carril, O., Eberhardt, J., Boltryk, S., Passecker, A., Thommen, B. T., Sifoniou, K., Scheurer, C., Fischli, C., Babai, D. I., Mahmoud, A. H., Alexander, L. T., Renner, S., Guiguemde, A. W., Pei, L., Gruering, C., Butendeich, H., Siebert, D., Straimer, J., Tobiasson, V., Baumgarten, S., Lill, M. A., Baeschlin, D. K., Rottmann, M., Voss, T. S., Brancucci, N. M. B.. 2026-09-13. A high-throughput compound screen identifies multiple druggable targets in Plasmodium falciparum transmission stages. https://doi.org/10.64898/2026.09.10.747198

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