Doxycycline has distinct apicoplast-specific mechanisms of antimalarial activity.

Doxycycline (DOX) is a key antimalarial drug thought to kill Plasmodium parasites by blocking protein translation in the essential apicoplast organelle. Clinical use is primarily limited to prophylaxis due to delayed second-cycle parasite death at 1-3 µM serum concentrations. DOX concentrations > 5 µM kill parasites with first-cycle activity but are thought to involve off-target mechanisms outside the apicoplast. We report that 10 µM DOX blocks apicoplast biogenesis in the first cycle and is rescued by isopentenyl pyrophosphate, an essential apicoplast product, confirming an apicoplast-specific mechanism. Exogenous iron rescues parasites and apicoplast biogenesis from first- but not second-cycle effects of 10 µM DOX, revealing that first-cycle activity involves a metal-dependent mechanism distinct from the delayed-death mechanism. These results critically expand the paradigm for understanding the fundamental antiparasitic mechanisms of DOX and suggest repurposing DOX as a faster acting antimalarial at higher dosing whose multiple mechanisms would be expected to limit parasite resistance.