Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

Abstract

Hematin crystallization is the primary mechanism of heme detoxification in malaria parasites and the target of the quinoline class of antimalarials. Despite numerous studies of malaria pathophysiology, fundamental questions regarding hematin growth and inhibition remain. Among them are the identity of the crystallization medium in vivo, aqueous or organic; the mechanism of crystallization, classical or nonclassical; and whether quinoline antimalarials inhibit crystallization by sequestering hematin in the solution, or by blocking surface sites crucial for growth. Here we use time-resolved in situ atomic force microscopy (AFM) and show that the lipid subphase in the parasite may be a preferred growth medium. We provide, to our knowledge, the first evidence of the molecular mechanisms of hematin crystallization and inhibition by chloroquine, a common quinoline antimalarial drug. AFM observations demonstrate that crystallization strictly follows a classical mechanism wherein new crystal layers are generated by 2D nucleation and grow by the attachment of solute molecules. We identify four classes of surface sites available for binding of potential drugs and propose respective mechanisms of drug action. Further studies reveal that chloroquine inhibits hematin crystallization by binding to molecularly flat {100} surfaces. A 2-μM concentration of chloroquine fully arrests layer generation and step advancement, which is ∼10 4 × less than hematin's physiological concentration. Our results suggest that adsorption at specific growth sites may be a general mode of hemozoin growth inhibition for the quinoline antimalarials. Because the atomic structures of the identified sites are known, this insight could advance the future design and/or optimization of new antimalarials. malaria parasites | heme detoxification | crystallization mechanisms | chloroquine | crystal growth inhibition

Early Onset of Kinetic Roughening due to a Finite Step Width in Hematin Crystallization

“…1(c)] [24,37]. The smooth surface agrees with the predictions of the above criteria for the interface structure of crystals near equilibrium with the solution.…”

“…We observed a smooth to rough transition [Figs. 1(c)-1(f)] during the growth of hematin crystals from a biomimetic mixed organic-aqueous solvent [23][24][25]; for experiment details, see Supplemental Material [26]. Hematin crystallization is the main pathway employed by malaria parasites to sequester toxic hematin, released during hemoglobin digestion [31]; its inhibition is considered the most successful target for antimalarial drugs [32].…”

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“…The formation of hemozoin is apparently the primary mechanism of heme detoxification in malaria parasites 12 . Free heme is oxidatively active and toxic to both the host cells and the malarial parasites, and it causes parasite death.…”

“…Free heme is oxidatively active and toxic to both the host cells and the malarial parasites, and it causes parasite death. Heme could cause extensive damage to membranes and inhibit a variety of enzymes resulting in the death of the parasite 12 .…”

Quinolines block every step of malaria heme crystal growth

“…In a previous paper, Olafson et al used citratebuffered saturated octanol to mimic an organic lipid environment to produce crystals monitored by atomic force microscopy (12). The evidence was a strictly classic mechanism of heme crystallization, with no evidence for a nonclassic incorporation of preformed hematin oligomers.…”