Tafenoquine Disrupts Intraerythrocytic Stages of Malaria Parasites by Inhibiting Nonclassical Pathways of β-Hematin Nucleation and Growth.

The rise in malaria resistance to multiple classes of drugs has led to an alarming increase in the number of cases and fatalities worldwide. Tafenoquine (TQ), an antimalarial approved by the Food and Drug Administration in 2018, inhibits multiple stages of malaria parasites' lifecycle, yet its precise mechanisms of action remain poorly understood. Here we explore how TQ interacts with the main pathway of heme detoxification in malaria parasites residing in erythrocytes: the formation of hemozoin crystals. We employ the synthetic analogue of hemozoin, β-hematin, and show that TQ blocks a fraction of the crystal growth sites and kinks and mildly inhibits the classical growth of β-hematin that occurs through the generation and spreading of layers. We demonstrate that TQ potently suppresses a nonclassical growth mode, which activates at elevated hematin concentrations as well as the nucleation of β-hematin crystals, by impeding the formation of mesoscopic hematin-rich clusters─the shared precursors of both crystal nucleation and nonclassical growth. Measurements of TQ parasite-killing potency reveal that, consistent with the mechanistic insights, this drug exerts a stage-specific inhibitory effect, with greater ring-stage suppression when hemozoin crystals nucleate than trophozoite suppression when the crystals grow. This mechanism highlights TQ's ability to disrupt parasite survival by inhibiting hemozoin synthesis in addition to the disruption of mitochondrial function.