(659g) Mechanism of Hematin Crystallization and Inhibition in Biomimetic Solutions

Crystallization is governed by thermodynamic and kinetic events that result in molecule (solute) incorporation into crystal surfaces. These fundamental processes mediate both natural and synthetic crystallization pathways. Our aim is to understand the molecular pathways for the crystallization of hematin, which is a byproduct of heme detoxification in malaria parasites. Upon entering its human host, the parasite catabolizes hemoglobin in red blood cells and releases free heme (Fe²⁺), which is rapidly oxidized to toxic hematin (Fe³⁺) within its digestive vacuole (DV). The parasite utilizes this process to sequester the toxin into innocuous crystals within the complex DV environment. Hematin crystal growth ensures parasite survival within the human host and is thought to be suppressed by quinolone-class antimalarial drugs. These crystals are referred to as hemozoin (in vivo) or β-hematin (in vitro). Identification of antimalarial drug action could provide a foundation for drug design to overcome the continued resurgence of antimalarial drug resistance. Here, we will discuss the molecular mechanism of hematin crystallization and inhibition by quinolone-class antimalarial drugs in a biomimetic growth environment.

In situ atomic force microscopy (AFM) revealed that hematin crystallization occurs through a classical layer-by-layer mechanism involving the addition of individual molecules to the growth sites present on the crystal surface.¹ Our studies employed a two-phase medium which we designed to mimic the complex environment of parasite DVs, which is comprised of an acidic aqueous phase and lipids. We used citric buffer-saturated octanol (CBSO) as a biomimetic solution to grow hematin crystals.² During in situ AFM experiments, we directly observe the effects of hematin surface diffusion and molecular level fluctuations in the rates of attachment/detachment from step edges. Towards understanding the relationship of solvent interactions on molecule addition, we quantified the rate of layer generation and the velocities of anisotropic step advancement as a function of supersaturation in the absence and presence of antimalarial modifiers. These quantifications provide a foundation for determining the modes of action of antimalarial drugs (i.e. inhibition of layer formation and/or step propagation). We identify that current antimalarials act by unique modes of inhibition. Here, we will summarize these findings and discuss our current studies to elucidate the role of functional moieties in current antimalarials and how the organic solvent affects drug-crystal interaction. To this end, we use a combination of in situ AFM and chemical force microscopy (CFM) measurements wherein AFM tips are functionalized with moieties that mimic specific regions of antimalarial drug molecules. Our findings reveal interesting trends of molecular recognition between antimalarial drugs and crystal surfaces that when coupled with bulk crystallization assays is a potential platform for the design and screening of new drug compounds for malaria.

References

1.  Olafson, K.N., M.A. Ketchum, J.D. Rimer, and P.G. Vekilov, Mechanisms of Hematin Crystallization and Inhibition by the Antimalarial Drug Chloroquine. Proceedings of the National Academy of Sciences U S A, 2015. 112: p. 4946-4951.

2.  Olafson, K.N., J.D. Rimer, and P.G. Vekilov, Growth of Large Hematin Crystals in Biomimetic Solutions. Crystal Growth & Design, 2014. 14(5): p. 2123-2127.