(603c) Recently Introduced Antimalarials Employ Nonclassical Mechanisms to Inhibit Hematin Crystallization

Malaria remains a significant public health challenge in equatorial regions of the world largely owing to the parasite's emerging resistance to current state-of-the-art drugs of the artemisinin (ART) family. In the human body most ART-derivative drugs are metabolized to dihydroartemisinin (DHA), which, in the parasite residing in the red blood cells, is activated by heme to form an adduct with hematin (H-DHA). In search of strategies to circumvent malaria parasites drug resistance, we explore the mechanism employed by H-DHA to inhibit the synthesis of hemozoin crystals, the main constituent of the heme detoxification pathway of the parasites. We also examine how tafenoquine (TQ), an antimalarial introduced in 2018, interacts with hemozoin formation. We show that both H-DHA and TQ block a fraction of the crystal growth sites, the kinks, and mildly inhibit the classical growth mode of b-hematin, the synthetic analogue of hemozoin, by generation and spreading of layers. H-DHA and TQ exert opposing effects on the nucleation of hematin crystals: H-DHA strongly enhances crystal nucleation, whereas TQ potently suppresses it. The two drugs accomplish their nucleation control missions by enhancing or impeding the formation of mesoscopic hematin-rich clusters, the precursors of hematin crystal nucleation. The diverging effects on crystal nucleation and the nucleation precursors trigger in two distinct nonclassical pathways to suppress hematin crystal growth that transform H-DHA and TQ into potent hematin growth inhibitors. With H-DHA we find that hematin crystallites, whose nucleation is promoted by H-DHA, incorporate into large hematin crystals and suppress their growth, likely by straining the crystal lattice. Importantly, the induced growth suppression is irreversible and persists even in the absence of H-DHA. TQ suppresses a nonclassical growth mode, which activates at elevated hematin concentrations, by impeding the formation of the hematin-rich clusters, the shared precursors of both crystal nucleation and nonclassical growth. Measurements of TQ parasite killing potency reveal that, consistently with the mechanistic insights, this drug exerts a stage-specific inhibitory effect and suppresses both ring stage parasites, when hemozoin crystals nucleate, and trophozoites, when the crystals grow. This dual-action mechanism highlights TQ’s ability to disrupt parasite survival at multiple points during the blood stage of infection.