Katy N. Olafson

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

Significance Approximately 40% of the global population is at risk for malaria infection and 300–660 million clinical episodes of Plasmodium falciparum malaria occur annually. During the malaria parasite lifecycle in human erythrocytes, heme released during hemoglobin catabolism is detoxified by sequestration into crystals. Many of the common antimalarials are believed to suppress the parasite by inhibiting hematin crystallization. We present, to our knowledge, the first evidence of the molecular mechanisms of hematin crystallization and antimalarial drug action as crystal growth inhibitors. These findings enable the identification and optimization of functional moieties that bind to crystal surface sites, thus providing unique guidelines for the discovery of novel antimalarials to combat increased parasite resistance to current drugs. 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 ∼104× 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.

Hematin crystallization from aqueous and organic solvents

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 parasites 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.

Growth of Large Hematin Crystals in Biomimetic Solutions

Hematin crystallization is an essential component of the physiology of malaria parasites. Several antimalarial drugs are believed to inhibit crystallization and expose the parasites to toxic soluble hematin. Hence, understanding the mechanisms of hematin crystal growth and inhibition is crucial for the design of new drugs. A major obstacle to microscopic, spectroscopic, and crystallographic studies of hematin crystallization has been the unavailability of large hematin crystals grown under conditions representative of the parasite anatomy. We have developed a biomimetic method to reproducibly grow large hematin crystals reaching 50 μm in length. We imitate the digestive vacuole of Plasmodium falciparum and employ a two-phase solution of octanol and citric buffer. The nucleation of seeds is enhanced at the interface between the aqueous and organic phases, where an ordered layer of octanol molecules is known to serve as substrate for nucleation. The seeds are transferred to hematin-saturated octanol in contact with citric buffer. We show that the crystals grow in the octanol layer, while the buffer supplies hydrogen ions needed for bonds that link the hematin molecules in the crystal. The availability of large hematin crystals opens new avenues for studies of hematin detoxification of malaria parasites in host erythrocytes.

Lipid or aqueous medium for hematin crystallization

Hematin crystallization is the primary heme detoxification mechanism of malaria parasites infecting human erythrocytes and the target of currently applied antimalarial medications. The composition of the crystallization medium within the parasites digestive vacuole (DV), the aqueous or lipid sub-phases, has been the subject of intense debate. Here we show that a blend of lipids, designed to mimic the lipid sub-phase in the parasite DV, contains significant amounts of soluble water that facilitates hematin crystal formation. We show that the hematin solubility in citric buffer-saturated n-octanol (CBSO), a model for the DV lipid sub-phase, is 100000-fold greater than in a biomimetic aqueous solution, indicating that organic-based crystallization provides an environment for faster hematin crystallization and more efficient heme detoxification. We demonstrate that hematin crystals grow with physiologically-relevant rates from CBSO and do not grow from our biomimetic aqueous solvents. Our findings suggest that hematin crystallization most likely requires the participation of lipid structures. We propose a mechanism of hematin crystallization in the parasite DV that reconciles this conclusion with data on hemoglobin transport and hematin generation and crystallization in vivo. The proposed mechanism suggests that hematin becomes incorporated into crystals via a layer of neutral lipids or possibly after penetrating the phospholipid bilayer of the DV. We specify guidelines for tests of the proposed mechanism and highlight its clinical and pharmacological implications.

Antimalarials inhibit hematin crystallization by unique drug–surface site interactions

Significance Approximately 3.2 billion people are at risk for malaria. The resistance of malaria parasites to current advanced multidrug treatments, recently recorded to spread out of Southeast Asia, has raised concerns of dire public health consequences. We demonstrate that antimalarial drugs suppress heme detoxification in the malaria parasites in a manner that is counter to the prevailing hypothesis in the field. We find that quinoline-class drugs work by specific interactions with β-hematin crystals, which are the by-product of heme detoxification within the digestive vacuole of the parasites. We also identify specific drug adsorption sites on crystal surfaces. These insights may potentially spur development of antimalarial drugs that overcome parasite resistance through a rational approach, far superior to currently used combinatorial methods. In malaria pathophysiology, divergent hypotheses on the inhibition of hematin crystallization posit that drugs act either by the sequestration of soluble hematin or their interaction with crystal surfaces. We use physiologically relevant, time-resolved in situ surface observations and show that quinoline antimalarials inhibit β-hematin crystal surfaces by three distinct modes of action: step pinning, kink blocking, and step bunch induction. Detailed experimental evidence of kink blocking validates classical theory and demonstrates that this mechanism is not the most effective inhibition pathway. Quinolines also form various complexes with soluble hematin, but complexation is insufficient to suppress heme detoxification and is a poor indicator of drug specificity. Collectively, our findings reveal the significance of drug–crystal interactions and open avenues for rationally designing antimalarial compounds.

Deconstructing Quinoline-Class Antimalarials to Identify Fundamental Physicochemical Properties of Beta-Hematin Crystal Growth Inhibitors

A versatile approach to control crystallization involves the use of modifiers, which are additives that interact with crystal surfaces and alter their growth rates. Elucidating a modifiers binding specificity to anisotropic crystal surfaces is a ubiquitous challenge that is critical to their design. In this study, we select hematin, a byproduct of malaria parasites, as a model system to examine the complementarity of modifiers (i.e., antimalarial drugs) to β-hematin crystal surfaces. We divide two antimalarials, chloroquine and amodiaquine, into segments consisting of a quinoline base, common to both drugs, and side chains that differentiate their modes of action. Using a combination of scanning probe microscopy, bulk crystallization, and analytical techniques, we show that the base and side chain work synergistically to reduce the rate of hematin crystallization. In contrast to general observations that modifiers retain their function upon segmentation, we show that the constituents do not act as modifiers. A systematic study of quinoline isomers and analogues shows how subtle rearrangement and removal of functional moieties can create effective constituents from previously ineffective modifiers, along with tuning their inhibitory modes of action. These findings highlight the importance of specific functional moieties in drug compounds, leading to an improved understanding of modifier-crystal interactions that could prove to be applicable to the design of new antimalarials.

Structuring of Organic Solvents at Solid Interfaces and Ramifications for Antimalarial Adsorption on β-Hematin Crystals

A critical aspect of material synthesis is solvent structuring at solid-liquid interfaces, which can impact the adsorption of solute and growth modifiers on an underlying substrate. In general, the impact of solvent structuring on molecular sorbate interactions with solid sorbents is poorly understood. This is particularly true for processes that occur in organic media, such as hematin crystallization, which is crucial to the survival of malaria parasites. Here, we use chemical force microscopy and molecular modeling to analyze the interactions between functional moieties of known antimalarials and the interface between β-hematin crystals and a mixed organic (octanol)-aqueous solvent. We show that the β-hematin surface, patterned in parallel hydrophobic and hydrophilic stripes, engenders the assembly of up to five layers of octanol molecules aligned parallel to the crystal surface. In contrast, studies of solvent structuring on a disordered glass surface reveal that octanol molecules align perpendicular to the interface. The distinct octanol arrays direct molecule adsorption at the respective interfaces. At both substrates, we also find stabilized pockets of aqueous nanophase lining the surfaces. A combination of experimental analyses and modeling of solvent structuring provides crucial insights into the association of hematin molecules with growing crystals as well as the adsorption and mobility of antimalarial drugs. Moreover, our findings offer a general perspective on the collective behaviors of complex organic solvents that may apply to a broad range of interactions at solid-liquid interfaces.

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

The structure of the interface of a growing crystal with its nutrient phase largely determines the growth dynamics. We demonstrate that hematin crystals, crucial for the survival of malaria parasites, transition from faceted to rough growth interfaces at increasing thermodynamic supersaturation Δμ. Contrary to theoretical predictions and previous observations, this transition occurs at moderate values of Δμ. Moreover, surface roughness varies nonmonotonically with Δμ, and the rate constant for rough growth is slower than that resulting from nucleation and spreading of layers. We attribute these unexpected behaviors to the dynamics of step growth dominated by surface diffusion and the loss of identity of nuclei separated by less than the step width w. We put forth a general criterion for the onset of kinetic roughening using w as a critical length scale.