Quinolines block every step of malaria heme crystal growth

Malaria is a lethal zoonotic disease that has impacted human survival and indeed, the history of human civilizations worldwide. The first effective treatment for malaria was reported in 1632 with the use of quinine extracts from the bark of the cinchona tree. Since that time, quinoline compounds have been used as both prophylactic and therapeutic drugs in every type of malarial treatment. Almost 400 y later, the molecular mechanism of quinoline action is brought into focus by Olafson et al. in their study of the step inhibition of heme crystal growth (1).

To put the significance of their findings into perspective, identification of hemozoin was integral to the 1880 malaria diagnosis by Laveran (2). Sometimes referred to as malaria pigment, this crystalline heme is synthesized by parasites intracellularly and was identified as a recognizable black round body with thin filaments on the periphery from exflagellation of the gametocytes in unstained human erythrocytes (2). Ronald Ross also found malarial pigment outside the Anopheles stomach to implicate the mosquito as the vector (3). In 1911, Brown (4) stated that hematin was a component of hemozoin, distinct from melanin. He also postulated malaria pigment was an impure byproduct of hemoglobin degradation. For the next 80 y, malariologists neglected hemozoin study until Slater and Cerami proved that hemozoin was pure heme by elemental analysis (5) and, importantly, that quinolines inhibited hemozoin growth (6).

Decades … 

[↵][1]1Email: dsulliv7{at}jhmi.edu.

 [1]: #xref-corresp-1-1

Malaria is a lethal zoonotic disease that has impacted human survival and indeed, the history of human civilizations worldwide. The first effective treatment for malaria was reported in 1632 with the use of quinine extracts from the bark of the cinchona tree. Since that time, quinoline compounds have been used as both prophylactic and therapeutic drugs in every type of malarial treatment. Almost 400 y later, the molecular mechanism of quinoline action is brought into focus by Olafson et al. in their study of the step inhibition of heme crystal growth (1).
To put the significance of their findings into perspective, identification of hemozoin was integral to the 1880 malaria diagnosis by Laveran (2). Sometimes referred to as malaria pigment, this crystalline heme is synthesized by parasites intracellularly and was identified as a recognizable black round body with thin filaments on the periphery from exflagellation of the gametocytes in unstained human erythrocytes (2). Ronald Ross also found malarial pigment outside the Anopheles stomach to implicate the mosquito as the vector (3). In 1911, Brown (4) stated that hematin was a component of hemozoin, distinct from melanin. He also postulated malaria pigment was an impure byproduct of hemoglobin degradation. For the next 80 y, malariologists neglected hemozoin study until Slater and Cerami proved that hemozoin was pure heme by elemental analysis (5) and, importantly, that quinolines inhibited hemozoin growth (6).
Decades ago, an initial hypothesis mentioned by Olafson et al. (1) concerning the mechanism of quinoline inhibition was binding to substrate heme to prevent incorporation into hemozoin. Cohen et al. first observed quinoline-heme complexes that were postulated to be toxic to the malaria parasites (7). Subsequent work demonstrated quinolone-heme complex interference with Plasmodium enzymes and insertion into membranes (8). However, this substrate hypothesis was not able to explain why many quinoline drugs that bind heme were unable to kill the malaria parasite. For example, the stereoisomer of quinine-9-epiquinine is an ineffective malaria drug (9). Egan et al. have proposed a hierarchy for structure-activity, which starts with heme binding, followed by heme crystal inhibition, as well as weak base accumulation in the acidic, lysosome-like specialized Plasmodium digestive vacuole designed for rapid hemoglobin 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 crystal growth accumulate at the target site to kill Plasmodium. Sullivan and Goldberg and their colleagues observed heme-quinoline complexes binding to hemozoin in situ by electron microscopy, subcellular fractionation, and in vitro with hemozoin extension assays, and proposed a quinoline-capping hemozoin growth mechanism (11). Unresolved were the diverse equilibriums between quinoline-heme complexes decreasing heme crystal substrate availability, heme substrate incorporation into heme crystal, heme and the mu-oxo-dimer, and quinolines binding to heme crystal.
In a previous paper, Olafson et al. used citratebuffered saturated octanol to mimic an organic lipid environment to produce crystals monitored by atomic force microscopy (12). The evidence was a strictly classic mechanism of heme crystallization, with no evidence for a nonclassic incorporation of preformed hematin oligomers. The authors described four classes of growth sites: (site 1) flat surfaces between steps, (site 2) nuclei of small numbers of heme dimers protruding from a flat face, (site 3) obtuse and acute kinks located on steps, and (site 4) groups of closely spaced steps, shown in Fig. 1. Bohle and colleagues (13), by powder diffraction, demonstrated head-to-tail heme dimers linking the proprionate carboxyl group to ferric iron of the adjacent heme, in which the pair are ∼1-nm square, as the fundamental building brick for heme crystal growth. The heme dimer 1-nm-square bricks then stack by hydrogen-bonding interactions into the crystal, which in Plasmodium falciparum are ∼100 nm × 100 nm × 500 nm. A neutral lipid nanosphere environment within the digestive vacuole excludes water, as well other interfering molecules, like amino acids or proteins in the diverse intracellular mixture (14). Kapishnikov and Leiserowitz and their colleagues applied crystallographic measures and soft Xray tomography to postulate a digestive vacuole membrane site of formation when examining late trophozoites (15)(16)(17). These authors also modeled quinolines binding to the surface of crystals (18). Hemozoin in early trophozoites has been observed to spin wildly in excess of Brownian motion in live parasites, which stop moving in drug-treated dead parasites (19,20). This finding may indicate that a neutral lipid environment within the digestive vacuole not attached to the digestive vacuole membrane exists for an early trophozoite portion of crystal formation.
In this new work, Olafson et al. (1) use atomic-force microscopy to capture the in situ growth of heme crystals to show how step propagation is inhibited by the quinoline class of antimalarials. The authors also observed the effects of artemisnin, another important antimalarial that binds heme, but does not inhibit crystallization (1). Direct observations of how these compounds affect growth lead Olafson et al. to detail three classes of quinolineinhibition mechanisms. First, the quinolones, amodiaquine and mefloquine, were found to largely only bind to kink growth sites where molecular units are added to a step edge by an inhibition mechanism that is termed "kink-blocking." This is the leasteffective mechanism of heme crystal growth inhibition. In a second mechanism, known as "step-pinning," the quinolines, such as chloroquine and quinine, bind anywhere on a flat surface face in addition to kink sites. By this process, heme crystal formation is inhibited over broad areas of the crystal surface. Finally, a single quinoline-tested pyronaridine inhibits growth by a step-bunching mechanism, whereby the ability to simultaneously bind at two step edges leads to a pile-up of the elementary steps into groups separated by large terraces.
Although not tested here, previous work indicated that a quinoline-heme complex binds more efficiently to the steps or kinks of hemozoin than quinolines alone (21,22). Quinolines can also form covalent bonds with heme to incorporate into the steps, but the proportion of covalent-bound quinoline-heme versus noncovalent-bound complexes has not been determined (23)(24)(25). At present, evidence points to a reversible quinoline-heme complex binding at the three types of sites rather than irreversible inhibition. This reversible binding is still governed by changes in pH associated with quinoline-resistant parasites or alterations in PfCRT or Pfmdr1 transporters associated with drug-resistant parasites. This work opens new opportunities for a lock-and-key approach to targeted drug development that is rooted in the physical basis for hemozoin crystal growth inhibition.