COMMENTARY

Quinolines block every step of malaria hemecrystal growthDavid J. Sullivan Jr.a,b,1

Malaria is a lethal zoonotic disease that has impactedhuman survival and indeed, the history of human civiliza-tions worldwide. The first effective treatment for malariawas reported in 1632 with the use of quinine extracts fromthe bark of the cinchona tree. Since that time, quinolinecompounds have been used as both prophylactic andtherapeutic drugs in every type of malarial treatment.Almost 400 y later, the molecular mechanism of quinolineaction is brought into focus by Olafson et al. in their studyof the step inhibition of heme crystal growth (1).

To put the significance of their findings into per-spective, identification of hemozoin was integralto the 1880 malaria diagnosis by Laveran (2). Some-times referred to as malaria pigment, this crystallineheme is synthesized by parasites intracellularly andwas identified as a recognizable black round bodywith thin filaments on the periphery from exflagella-tion of the gametocytes in unstained human erythro-cytes (2). Ronald Ross also found malarial pigmentoutside the Anopheles stomach to implicate the mos-quito as the vector (3). In 1911, Brown (4) stated thathematin was a component of hemozoin, distinct frommelanin. He also postulated malaria pigment was animpure byproduct of hemoglobin degradation. Forthe next 80 y, malariologists neglected hemozoinstudy until Slater and Cerami proved that hemozoinwas pure heme by elemental analysis (5) and, impor-tantly, that quinolines inhibited hemozoin growth (6).

Decades ago, an initial hypothesis mentioned byOlafson et al. (1) concerning the mechanism of quinolineinhibition was binding to substrate heme to prevent in-corporation into hemozoin. Cohen et al. first observedquinoline–heme complexes that were postulated to betoxic to the malaria parasites (7). Subsequent work dem-onstrated quinolone–heme complex interference withPlasmodium enzymes and insertion into membranes(8). However, this substrate hypothesis was not ableto explain why many quinoline drugs that bind hemewere unable to kill the malaria parasite. For example,the stereoisomer of quinine-9-epiquinine is an ineffectivemalaria drug (9). Egan et al. have proposed a hierarchyfor structure-activity, which starts with heme binding, fol-lowed by heme crystal inhibition, as well as weak base

accumulation in the acidic, lysosome-like specializedPlasmodium digestive vacuole designed for rapid he-moglobin digestion and heme crystal formation (10).Not all drugs that bind heme stop heme crystal growth,nor do all drugs that bind heme and inhibit heme crystalgrowth accumulate at the target site to kill Plasmodium.Sullivan and Goldberg and their colleagues observedheme–quinoline complexes binding to hemozoin in situby electron microscopy, subcellular fractionation, andin vitro with hemozoin extension assays, and proposeda quinoline-capping hemozoin growth mechanism (11).Unresolved were the diverse equilibriums betweenquinoline–heme complexes decreasing heme crystalsubstrate availability, heme substrate incorporationinto heme crystal, heme and the mu-oxo–dimer, andquinolines binding to heme crystal.

In a previous paper, Olafson et al. used citrate-buffered saturated octanol to mimic an organic lipidenvironment to produce crystals monitored by atomicforce microscopy (12). The evidence was a strictly classicmechanism of heme crystallization, with no evidence fora nonclassic incorporation of preformed hematin oligo-mers. The authors described four classes of growth sites:(site 1) flat surfaces between steps, (site 2) nuclei of smallnumbers of heme dimers protruding from a flat face, (site3) obtuse and acute kinks located on steps, and (site 4)groups of closely spaced steps, shown in Fig. 1. Bohleand colleagues (13), by powder diffraction, demon-strated head-to-tail heme dimers linking the proprionatecarboxyl group to ferric iron of the adjacent heme, inwhich the pair are ∼1-nm square, as the fundamentalbuilding brick for heme crystal growth. The heme dimer1-nm-square bricks then stack by hydrogen-bonding in-teractions into the crystal, which in Plasmodium falcipa-rum are ∼100 nm × 100 nm × 500 nm. A neutral lipidnanosphere environment within the digestive vacuoleexcludes water, as well other interfering molecules, likeamino acids or proteins in the diverse intracellular mix-ture (14). Kapishnikov and Leiserowitz and their col-leagues applied crystallographic measures and soft X-ray tomography to postulate a digestive vacuole mem-brane site of formationwhen examining late trophozoites(15–17). These authors also modeled quinolines binding

aDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205;and bDepartment of Medicine, Division of Infectious Diseases, The Johns Hopkins School of Medicine, Baltimore, MD 21205Author contributions: D.J.S. wrote the paper.The author declares no conflict of interest.See companion article on page 7531.1Email: dsulliv7@jhmi.edu.

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to the surface of crystals (18). Hemozoin in early trophozoites hasbeen observed to spin wildly in excess of Brownian motion in liveparasites, which stop moving in drug-treated dead parasites (19, 20).This finding may indicate that a neutral lipid environment within thedigestive vacuole not attached to the digestive vacuole membraneexists for an early trophozoite portion of crystal formation.

In this new work, Olafson et al. (1) use atomic-force microscopyto capture the in situ growth of heme crystals to show how steppropagation is inhibited by the quinoline class of antimalarials.The authors also observed the effects of artemisnin, another im-portant antimalarial that binds heme, but does not inhibit crystal-lization (1). Direct observations of how these compounds affectgrowth lead Olafson et al. to detail three classes of quinoline-inhibition mechanisms. First, the quinolones, amodiaquine andmefloquine, were found to largely only bind to kink growth siteswhere molecular units are added to a step edge by an inhibitionmechanism that is termed “kink-blocking.” This is the least-effective mechanism of heme crystal growth inhibition. In a sec-ond mechanism, known as “step-pinning,” the quinolines, such aschloroquine and quinine, bind anywhere on a flat surface face in

addition to kink sites. By this process, heme crystal formation isinhibited over broad areas of the crystal surface. Finally, a singlequinoline-tested pyronaridine inhibits growth by a step-bunchingmechanism, whereby the ability to simultaneously bind at twostep edges leads to a pile-up of the elementary steps into groupsseparated by large terraces.

Although not tested here, previous work indicated that aquinoline–heme complex binds more efficiently to the steps orkinks of hemozoin than quinolines alone (21, 22). Quinolines canalso form covalent bonds with heme to incorporate into the steps,but the proportion of covalent-bound quinoline–heme versusnoncovalent-bound complexes has not been determined (23–25). At present, evidence points to a reversible quinoline–hemecomplex binding at the three types of sites rather than irreversibleinhibition. This reversible binding is still governed by changes inpH associated with quinoline-resistant parasites or alterations inPfCRT or Pfmdr1 transporters associated with drug-resistant par-asites. This work opens new opportunities for a lock-and-key ap-proach to targeted drug development that is rooted in thephysical basis for hemozoin crystal growth inhibition.

Fig. 1. Quinoline blockade of heme crystal growth by step-pinning or kink-blocking. Head-to-tail heme dimers add in a classic crystallizationmechanism at growth sites, which are: (1) terraces or open facers, (2) nuclei of a few heme dimers, (3) kink sites, and (4) closely spaced steps.Quinolines block by open flat face attachment in the instance of chloroquine and quinine, kink-blocking in the case of amodiaquine andmefloquine, and by step-bunching in the case of pyronaridine.

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1 Olafson KN, Nguyen TQ, Rimer JD, Vekilov PG (2017) Antimalarials inhibit hematin crystallization by unique drug–surface site interactions. Proc Natl Acad Sci USA114:7531–7536.

2 Laveran A (1880) Un nouveau parasite trouve dans le sang des malade atteints de fievre palustre. Origine parasitaire des accidents de l’impaludisme. Bull MemSoc Med Hop Paris 7:158–164.

3 Ross R (1897) On some peculiar pigmented cells found in two mosquitos fed on malarial blood. BMJ 2:1786–1788.4 Brown WH (1911) Malarial pigment (so-called melanin): Its nature and mode of production. J Exp Med 13:290–299.5 Slater AF, et al. (1991) An iron-carboxylate bond links the heme units of malaria pigment. Proc Natl Acad Sci USA 88:325–329.6 Slater AF, Cerami A (1992) Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites. Nature 355:167–169.7 Cohen SN, Phifer KO, Yielding KL (1964) Complex formation between chloroquine and ferrihaemic acid in vitro, and its effect on the antimalarial action ofchloroquine. Nature 202:805–806.

8 Orjih AU, Banyal HS, Chevli R, Fitch CD (1981) Hemin lyses malaria parasites. Science 214:667–669.9 Slater AF (1993) Chloroquine: Mechanism of drug action and resistance in Plasmodium falciparum. Pharmacol Ther 57:203–235.

10 Egan TJ, et al. (2000) Structure-function relationships in aminoquinolines: Effect of amino and chloro groups on quinoline-hematin complex formation, inhibitionof beta-hematin formation, and antiplasmodial activity. J Med Chem 43:283–291.

11 Sullivan DJ, Jr, Gluzman IY, Russell DG, Goldberg DE (1996) On the molecular mechanism of chloroquine’s antimalarial action. Proc Natl Acad Sci USA93:11865–11870.

12 Olafson KN, Ketchum MA, Rimer JD, Vekilov PG (2015) Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine. Proc Natl AcadSci USA 112:4946–4951.

13 Pagola S, Stephens PW, Bohle DS, Kosar AD, Madsen SK (2000) The structure of malaria pigment beta-haematin. Nature 404:307–310.14 Pisciotta JM, et al. (2007) The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization. Biochem J 402:197–204.15 Kapishnikov S, et al. (2013) Digestive vacuole membrane in Plasmodium falciparum-infected erythrocytes: Relevance to templated nucleation of hemozoin.

Langmuir 29:14595–14602.16 Kapishnikov S, et al. (2012) Oriented nucleation of hemozoin at the digestive vacuole membrane in Plasmodium falciparum. Proc Natl Acad Sci USA

109:11188–11193.17 Kapishnikov S, et al. (2012) Aligned hemozoin crystals in curved clusters in malarial red blood cells revealed by nanoprobe X-ray Fe fluorescence and diffraction.

Proc Natl Acad Sci USA 109:11184–11187.18 Solomonov I, et al. (2007) Crystal nucleation, growth, and morphology of the synthetic malaria pigment beta-hematin and the effect thereon by quinoline

additives: The malaria pigment as a target of various antimalarial drugs. J Am Chem Soc 129:2615–2627.19 Sachanonta N, et al. (2011) Ultrastructural and real-time microscopic changes in P. falciparum-infected red blood cells following treatment with antimalarial drugs.

Ultrastruct Pathol 35:214–225.20 Sigala PA, Goldberg DE (2014) The peculiarities and paradoxes of Plasmodium heme metabolism. Annu Rev Microbiol 68:259–278.21 Sullivan DJ, Jr, Matile H, Ridley RG, Goldberg DE (1998) A common mechanism for blockade of heme polymerization by antimalarial quinolines. J Biol Chem

273:31103–31107.22 Iyer JK, Shi L, Shankar AH, Sullivan DJ, Jr (2003) Zinc protoporphyrin IX binds heme crystals to inhibit the process of crystallization in Plasmodium falciparum.Mol

Med 9:175–182.23 Alumasa JN, et al. (2011) The hydroxyl functionality and a rigid proximal N are required for forming a novel non-covalent quinine-heme complex. J Inorg Biochem

105:467–475.24 Gorka AP, de Dios A, Roepe PD (2013) Quinoline drug-heme interactions and implications for antimalarial cytostatic versus cytocidal activities. J Med Chem

56:5231–5246.25 Gorka AP, Sherlach KS, de Dios AC, Roepe PD (2013) Relative to quinine and quinidine, their 9-epimers exhibit decreased cytostatic activity and altered heme

binding but similar cytocidal activity versus Plasmodium falciparum. Antimicrob Agents Chemother 57:365–374.

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