Fungal-derived methyldeoxaphomins target <i>Plasmodium falciparum</i> segregation through the inhibition of PfActin1

Herein we report the finding and structure determination of a natural product based on the methyldeoxaphomin scaffold family from the fungus <i>Trichocladium asperum</i> that shows promising antiplasmodial activity and selectivity against host cells. In vitro evolution and whole genome analysis in <i>Plasmodium falciparum</i> with the most potent member, NPDG-F (EC₅₀ of 550 nM in Dd2; 290 nM in 3D7), shows that parasite resistance to methyldeoxaphomins is strongly associated with mutations in PfActin1 (PF3D7_1246200), a critically essential ATPase needed for all stages of parasite development. Molecular docking study with available PfActin1 crystal structure shows NPDG-F occupies the same allosteric binding pocket as the known actin inhibitor cytochalasin D. The direct PfActin1 target engagement in the allosteric site was supported by cross-resistance studies, isobologram analysis with other PfActin1 inhibitors, and the structure-activity relationships for the methyldeoxaphomin family. When added to in vitro culture, NPDG-F induced morphological abnormalities in merozoite cellularization during schizogony in both the <i>Plasmodium</i> blood and liver stages. Our data provide chemical validation that PfActin1 is an attractive, pan-lifecycle target and inform strategies for the design of more selective inhibitors.