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

Hematin crystallization is the main mechanism of detoxification of heme that is released in malaria-infected erythrocytes as a byproduct of the hemoglobin catabolism by the parasite. A controversy exists over whether hematin crystals grow from the aqueous medium of the parasite's digestive vacuole or in the lipid bodies present in the vacuole. To this end, we compare the basic thermodynamic and structural features of hematin crystallization in an aqueous buffer at pH 4.8, as in the digestive vacuole, and in water-saturated octanol that mimics the environment of the lipid nanospheres. We show that in aqueous solutions, hematin aggregation into mesoscopic disordered clusters is insignificant. We determine the solubility of the β-hematin crystals in the pH range 4.8-7.6. We image by atomic force microscopy crystals grown at pH 4.8 and show that their macroscopic and mesoscopic morphology features are incompatible with those reported for biological hemozoin. In contrast, crystals grown in the presence of octanol are very similar to those extracted from parasites. We determine the hematin solubility in water-saturated octanol at three temperatures. These solubilities are four orders of magnitude higher than that at pH 4.8, providing for faster crystallization from organic than from aqueous solvents. These observations further suggest that the lipid bodies play a role in mediating biological hemozoin crystal growth to ensure faster heme detoxification.

Here, we employ n-octanol to elucidate the fundamental processes of hematin crystallization from an organic solvent, identify the operational mechanisms of growth, and determine the respective control parameters. The values of the enthalpy, entropy, and free energy of the crystal−solution equilibrium suggest that octanol may structure around the hematin solute molecules and along the crystal interface. Time-resolved in situ atomic force microscopy demonstrates that hematin crystal growth strictly follows classical layer growth mechanisms. Steps propagate by the attachment of solute molecules, described by a first-order chemical rate law. The molecules reach the steps via adsorption on the crystal surface, followed by surface diffusion, and the kinetic barriers of this pathway offer additional crystallization control strategies. Solute incorporation into steps from the adjacent lower and upper terraces is strongly asymmetric, with the lower terrace contributing the major solute amount. These findings provide a foundation for the rational design of hematin crystals that may find applications utilizing their high magnetic and optical anisotropy.

Hematin crystallization, the primary heme detoxification mechanism of malaria parasites infecting human erythrocytes, most likely requires the participation of lipid structures.

Molecular inhibitors are commonly employed in natural, synthetic, and biological crystallization as a means of regulating crystal size and habit. In pathological crystallization, growth inhibitors are commonly employed as therapies to decrease the rate of crystal growth, thereby reducing the incidence rate of diseases. When designing inhibitors for such purposes, it is often unknown a priori what properties (e.g., structure, functionality) are critical for drug efficacy and specificity. Identification of lead candidates is often accomplished through brute force screening without knowledge of the molecular-level driving force(s) governing favorable inhibitor− crystal interactions. Here, we present a biomimetic assay to characterize the crystallization of hematin, a critical component of Plasmodium parasite survival in malaria. Many antimalarial drugs have been shown to function as inhibitors that suppress hematin crystal growth. The increasing resistance of malaria parasites to current antimalarials has created an impetus to design alternative drugs; however, current approaches to identify lead candidates rely on combinatorial methods employing parasite assays to screen compounds, which are incapable of elucidating the effect(s) of inhibitors on hematin crystallization. In this study, we use citric-buffer saturated octanol (CBSO) as a physiologically relevant growth medium to develop a facile, robust method of screening compounds that inhibit hematin crystal growth. As benchmarks, we selected antimalarials and antibiotics that are commonly used in combination therapies. We assessed drug solubility in CBSO and designed a biomimetic assay to quantify the relative efficiency of hematin growth inhibitors. We demonstrate that this assay can be used as a high-throughput platform to screen libraries of compounds as a more streamlined approach to identify inhibitors of pathological crystallization.