The Neutral Lipid Composition Present in the Digestive Vacuole of <i>Plasmodium falciparum</i> Concentrates Heme and Mediates β-Hematin Formation with an Unusually Low Activation Energy

Hoang, Anh; Sandlin, Rebecca D.; Omar, A. Saldarriaga; Egan, Timothy J.; Wright, David W.

doi:10.1021/bi101397u

Cited by 60 publications

(87 citation statements)

References 52 publications

(111 reference statements)

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“…Hemozoin crystals have been observed immersed in the lipid nanospheres (9,16), or with their basal surfaces attached to the DV membrane (18)(19)(20) and other crystal faces apparently exposed to the aqueous subphase. Whereas it has been hypothesized that hemozoin crystals nucleate on the DV membrane (17,20), the debate on the medium, aqueous or organic, from which soluble hematin reaches the crystals and associates to them has yet to be reconciled (18)(19)(20)(31)(32)(33).…”

Section: Resultsmentioning

confidence: 99%

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

Olafson

Ketchum

Rimer

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

151

192

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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

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Section: Resultsmentioning

confidence: 99%

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

Olafson

Ketchum

Rimer

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

151

192

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“…Neutral lipids are required to support membrane biogenesis, and are also known to play a role in the phenomenon of haemozoin formation in the malaria parasite's DV 30 . ABCG2, in mammals, protects against toxic levels of the haeme precursor protoporphyrin IX (ref.…”

Section: Discussionmentioning

confidence: 99%

A female gametocyte-specific ABC transporter plays a role in lipid metabolism in the malaria parasite

et al. 2014

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ATP-binding cassette (ABC) transporters serve a variety of physiological functions as well as play key roles in drug resistance. The genome of the human malaria parasite, Plasmodium falciparum, encodes multiple members of this family, one of which, gABCG2, is transcribed predominantly in the gametocyte stage. Here we use gene deletion and tagging to investigate the expression, localization and function of gABCG2. The protein is found in a single dot-like lipid-rich structure within female, but not male, gametocytes. gABCG2-knockout cell lines produce more gametocytes of both sexes. By contrast, cholesteryl esters, diacylglycerols and triacylglycerols are significantly reduced in gABCG2-knockout gametocyte stages. We propose a role for gABCG2 in the regulation of gametocyte numbers and in the accumulation of neutral lipids, which are likely important for parasite development in the insect stages of the parasite life cycle.

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“…In vivo formation of hemozoin is likely mediated by neutral lipid particles located in the acidic digestive vacuole of the parasite (pH 4.9), where hemoglobin digestion occurs (4,23). Indeed, a recent study demonstrated that these lipids are kinetically sufficient mediators of ␤-hematin formation and could account for the detoxification of free heme in vivo (13). Unfortunately, the relatively high cost associated with neutral lipids and the requirement to prepare fresh mediator before each screen limits their use in HTS.…”

Section: Discussionmentioning

confidence: 99%

“…This solubilization of free heme has been shown to be the rate-limiting step in this reaction. It is doubtful that the in vivo process of hemozoin formation occurs through a similar mechanism of solubilization (6,13). In the NP-40 detergent assay, however, the detergent micelles more accurately mimic the neutral lipid particles present within the native environment of the parasite.…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of these lipid particles by mass spectrometry identified a specific blend of monostearic, monopalmitic, dipalmitic, dioleic, and dilinoleic glycerols in a 4:2:1: 1:1 ratio (23). In vitro studies have confirmed that this ratio of lipids is sufficient for mediation of ␤-hematin, synthetic hemozoin, under physiological conditions at a biologically realistic rate (12,13).…”

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confidence: 89%

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Use of the NP-40 Detergent-Mediated Assay in Discovery of Inhibitors of β-Hematin Crystallization

Sandlin

Carter

Lee

et al. 2011

Antimicrob Agents Chemother

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The protozoan parasite responsible for malaria affects over 500 million people each year. Current antimalarials have experienced decreased efficacy due to the development of drug-resistant strains of Plasmodium spp., resulting in a critical need for the discovery of new antimalarials. Hemozoin, a crystalline by-product of heme detoxification that is necessary for parasite survival, serves as an important drug target. The quinoline antimalarials, including amodiaquine and chloroquine, act by inhibiting the formation of hemozoin. The formation of this crystal does not occur spontaneously, and recent evidence suggests crystallization occurs in the presence of neutral lipid particles located in the acidic digestive vacuole of the parasite. To mimic these conditions, the lipophilic detergent NP-40 has previously been shown to successfully mediate the formation of ␤-hematin, synthetic hemozoin. Here, an NP-40 detergent-based assay was successfully adapted for use as a high-throughput screen to identify inhibitors of ␤-hematin formation. The resulting assay exhibited a favorable Z of 0.82 and maximal drift of less than 4%. The assay was used in a pilot screen of 38,400 diverse compounds at a screening concentration of 19.3 M, resulting in the identification of 161 previously unreported ␤-hematin inhibitors. Of these, 48 also exhibited >90% inhibition of parasitemia in a Plasmodium falciparum whole-cell assay at a screening concentration of 23 M. Eight of these compounds were identified to have nanomolar 50% inhibitory concentration values near that of chloroquine in this assay.

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The Neutral Lipid Composition Present in the Digestive Vacuole of Plasmodium falciparum Concentrates Heme and Mediates β-Hematin Formation with an Unusually Low Activation Energy

Cited by 60 publications

References 52 publications

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

A female gametocyte-specific ABC transporter plays a role in lipid metabolism in the malaria parasite

Use of the NP-40 Detergent-Mediated Assay in Discovery of Inhibitors of β-Hematin Crystallization

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