Interplay Between Malaria, Crystalline Hemozoin Formation, and

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Chem. Rev. 2008, 108, 4899–4914

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Interplay Between Malaria, Crystalline Hemozoin Formation, and Antimalarial Drug Action and Design Isabelle Weissbuch* and Leslie Leiserowitz* Department of Materials and Interfaces, The Weizmann Institute of Science, 76100-Rehovot, Israel Received January 22, 2008

Contents 1. Introduction 2. Crystal Structure and Morphology of Synthetic (β-Hematin) and Natural Hemozoin 2.1. Introduction 2.2. The Link between Crystal Structure and Morphology of β-Hematin 2.2.1. General Principles 2.2.2. Theoretical and Observed Morphology of β-Hematin and Hemozoin 3. The Effect of Antimalarial Drugs on Growth of β-Hematin and Hemozoin 3.1. Introduction 3.2. Mode of Action of Antimalarial Quinoline Drugs 3.2.1. Proposed Binding Sites of Quinoline Antimalarials on Hemozoin Crystal Faces 3.2.2. Experimental Evidence of Quinoline Binding to Hemozoin and β-Hematin Crystal Surfaces 3.3. Hemozoin as a Target of Antimalarials Containing Two Amino-Terminating Chains 3.3.1. The 3,6-Bis-ω-diethylamino-alkoxyxanthone Series 3.4. The Arteminsinin Type Drugs as Tailor-Made Inhibitor of Hemozoin Growth 3.5. Rationale for Antimalarial Drug Design with Hemozoin Crystal Surfaces as a Target 3.5.1. Design of Hemozoin Surface CMOLs for Growth Inhibition 3.5.2. Hemozoin Growth Inhibitors as TMAs that Resemble the β-Hematin Molecular Dimer 3.5.3. Antimalarial Combination Therapy Involving Artemisinin and Quinoline Type Drugs 4. Nucleation of Hemozoin Crystals 5. Summary 6. Acknowledgments 7. References

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1. Introduction This review on the interplay between malaria, antimalarial drugs, and hemozoin crystals is directed, on one hand, to biologists and pharmaceutical chemists who wish to have a * To whom correspondence should be E-mail: (I.W.) [email protected] [email protected].

addressed. or (L.L.)

The review represents the fruits, the offshoot, one may say, that may be reaped from the experience and knowledge gleaned from a collaborative effort, extending for more than a quarter of a century, on the design and use of auxiliaries for the control of crystal nucleation, morphology, and polymorphism, the structure determination of mono- and multilayer films of amphiphilic molecules by grazing incidence synchrotron X-ray diffraction at the air-liquid interface, and computational studies of molecular interactions at interfaces and in the crystal bulk. These studies have been conducted in close collaboration with Meir Lahav and with other colleagues.

broader knowledge of the crystalline state related to malaria and, on the other hand, to crystallographers and physical chemists who wish to have a simple introduction to the disease and drugs employed against it. The call for this review is dictated by various considerations, not the least being the need for new antimalarial drugs, in view of developing parasitic resistance to the commonly used ones.1 The increasing spread of malaria is also due to several other contributing factors; besides climatic and environmental factors, the Anopheles mosquito has become increasingly resistant to insecticides and has adapted so as to avoid

10.1021/cr078274t CCC: $71.00  2008 American Chemical Society Published on Web 11/12/2008

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Figure 1. Geographic distribution of malaria in the world. Reproduced with permission from the CDC Centers for Disease Control and Prevention Website.

insecticide-treated surfaces. Human malaria, a tropical, reemerging infectious disease caused by several types of protozoan parasites of the genus Plasmodium, has been a primary concern to humanity for centuries and is now extended to more than 40% of the world’s population. It is a disease primarily of the tropics but is also found in many temperate regions of the world including parts of the Middle East and Asia (Figure 1). The disease is responsible for some 300 million cases resulting in 1-3 million deaths per year, being particularly fatal among children.2 Humans are affected by five protozoa parasitic types of the genus Plasmodium (P.): P. falciparum, P. ViVax, P. oVale, P. malariae, and occasionally P. knowlesi. Of these, falciparum, the most prevalent species across the globe, is often fatal to humans. The life cycle of the malaria parasite is complex and incompletely understood. During the course of its life cycle, the parasite resides in both the human host and the mosquito vector at various developmental stages and forms, as depicted and outlined in Figure 2. One of the most extensively studied stages is that of the red blood cell (RBC), in which the parasite digests hemoglobin for its source of amino acids and iron. The parasite can digest up to 75% of the RBC’s content in a process whereby the globin is enzymatically cleaved into small peptides, producing free heme [Fe2+-protoporphyrin IX (Fe2+-PPIX)], as a byproduct that is toxic to the parasite. This heme undergoes a one-electron oxidation to produce ferriprotoporphyrin, Fe3+-PPIX (Figure 3a), that precipitates in the digestive food vacuole of the parasite as submicrometer to micrometer-sized crystals, known as hemozoin (Figure 4), which are harmless to the parasite. Hemozoin, the malaria pigment, is a marker of malaria infection in RBC assays. This process of hemozoin crystallization is now being considered as a type of biomineralization,3-5 although from the viewpoint of the human host, pathological crystallization is perhaps a more appropriate term. As alluded to above, a primary cause for the reemergence of malaria is a consequence of developing parasite resistance to antimalarial drugs, in particular some of the widest used synthetic quinolines. These antimalarials are known to act during the degradation of hemoglobin and subsequent production of malaria pigment crystals in the RBC. Moreover, there is some reason to believe that the modern antimalarial drug artemisinin and synthetic analogues thereof act in a similar manner. This review will concentrate on the RBC stage, where the focus will be on the crystallization and morphology of natural and synthetic hemozoin, crystal morphology, and growth inhibition via antimalarials, as well as how such drugs may best be designed.

Figure 2. Life cycle of the malaria parasite in humans and the mosquito vector. The parasite, in the form of sporozoites, is transmitted to humans during a blood meal by Anopheles mosquitoes. They pass within an hour into the liver, where they develop into schizonts. After 5-15 days, the schizonts then rupture, each releasing several thousand “daughter” parasites called merozoites into the blood stream, where they invade red blood cells (RBCs). Each such merozoite matures into a schizont containing several merozoites inside the RBCs, eventually rupturing to release the merozoites, which are then free to infect other RBCs. This process is responsible for the symptoms and pathological characteristics of the disease. Some merozoites differentiate into male and female forms, gametocytes, which are sucked up by a mosquito during its blood meal. Various transformations take place, eventually resulting in sporozoites that are liberated into the mosquito salivary glands, from where they may be injected back into the human host. Reprinted with permission from ref 6 (Malaria: Obstacles and Opportunities). Copyright 1991 National Academy Press.

A comprehensive overview of the obstacles and opportunities in the war against malaria has been published in book form, albeit 16 years ago.6 Recently, a book comprising articles on drug action and resistance and clinical and biological aspects of the disease has been published, within which of particular relevance here are reviews by Bray et al. on the mode of action of quinoline-containing drugs and recently developed resistance to commonly used quinolines by malaria parasites,7 by Sullivan et al. on bioavailable iron and heme metabolism in P. falciparum,8 and by Goldberg on hemoglobin degradation.9 Other recent reviews, describing established and new antimalarial drugs in clinical use and development10 and hemozoin as a primary target of antimalarials,11 are of relevance here.

2. Crystal Structure and Morphology of Synthetic (β-Hematin) and Natural Hemozoin 2.1. Introduction β-Hematin, the synthetic form of hemozoin, was believed in the 1990s to consist of strands of FeIII-porphyrin units

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growth and result in a build up of the toxic heme and thus the death of the parasite. This prediction was reinforced by in vitro experiments,3,15,17-21 using the incubation of hemozoin crystals and its analogues with various compounds from the quinoline family found to retard the crystal growth. No information, however, was provided by Bohle et al.13 on the crystal surface binding sites or details on the habit (i.e., overall shape) in terms of the crystalline axes and form (i.e., the {h,k,l} faces) of the micrometer-sized hemozoin or β-hematin crystals. Such knowledge of the crystal morphology (i.e., habit and form) is necessary to determine possible antimalarial drug binding sites and how they would affect the growth and morphology of synthetic or biogenic malaria pigment crystals. Figure 3. (a) Heme molecular structure. (b) The packing arrangement of β-hematin, which consists of molecular dimers, viewed along the c-axis.29 The reported13 crystal structure has cell constants a ) 12.20 Å, b ) 14.68 Å, c ) 8.04 Å, R ) 90.2°, β ) 96.8°, γ ) 97.9°, and space group P1j.

Figure 4. Schematic presentation of a RBC infected with a malaria parasite, whose digestive vacuole is shown containing mature hemozoin (HZ) crystals.

interlinked into a polymer via propionate oxygen-iron bonds.12 In a recent landmark study, Pagola et al. determined the crystal structure of β-hematin by powder X-ray diffraction (PXRD) of micrometer-sized crystals using synchrotron radiation.13 The authors showed the crystal structure to be centrosymmetric, space group P1j, consisting of propionatelinked [Fe-O-C(dO)CH2CH2-] reciprocal head-to-tail cyclichemedimersinterlinkedvia(propionicacid)O-H · · · OdC hydrogen-bonded cyclic pairs forming chains aligned parallel to the axial direction a-c (Figure 3b). It is noteworthy that the crystal structure of β-hematin gave a satisfactory fit to the PXRD patterns of hemozoin produced in the blood fluke Schistosoma mansoni and the kissing bug Rhodnius prolixus.14 The crystal structure of β-hematin led Bohle et al.13 to propose, in keeping with previous similar suggestions,15,16 when it was still believed that hemozoin consists of polymeric strands, that antimalarial quinoline drugs act by binding to hemozoin crystal faces, which would inhibit their

2.2. The Link between Crystal Structure and Morphology of β-Hematin 2.2.1. General Principles The morphology of a crystal is determined by the relative growth rates of its various faces, the general rule being that faces that grow slowest are expressed in the crystal habit. The basic rules for a quantitative determination of crystal morphology as determined by the crystal structure only were laid down by Hartman and Perdok,22,23 Hartman,24 and Hartman and Bennema.25 The crucial relation is between the layer energy, El, the energy released when a layer is formed, and the attachment energy, Eatt, the energy per molecule released when a new layer is attached to the crystal face. Eatt controls the growth rate perpendicular to the layer, whereas El measures the stability of the layer. The morphological importance of a crystal face, namely, its area, is inversely proportional to its attachment energy relative to that of the other faces. The energies El and Eatt may be computed using atom-atom potential energy functions, given the crystal structure. By applying such a procedure, it became possible to calculate the energy values of El and Eatt for various low-index (h,k,l) crystal faces, from which the “theoretical crystal form” may be derived. This method has been applied for a variety of organic and inorganic crystals. It is important to note that solvent and additives can dramatically affect the crystal habit, as well as the type of faces expressed.

2.2.2. Theoretical and Observed Morphology of β-Hematin and Hemozoin The computed crystal attachment energies Eatt reported by Buller et al.26 yielded the theoretical growth form of β-hematin (Figure 5a,b). The crystal morphology is needlelike, extending along the c-axis, exhibiting dominant {100} and {010} side faces, a less-developed {011}, and a minor {001} facet. Faces of the type {hkl}, where h, k, and l are nonzero (or have high hkl indices), are not formed since they have significant negative attachment energies, in keeping with the highly corrugated nature of such faces. By a similar criterion, the {hk0} side faces of the needlelike crystal of β-hematin, such as {(110}, are not manifest, being corrugated and thus with attachment energies more negative than either {100} or {010} faces (Figure 6a). Of the (0kl} faces, only the {011} appears in the theoretical form, as opposed

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3. The Effect of Antimalarial Drugs on Growth of β-Hematin and Hemozoin 3.1. Introduction

Figure 5. (a,b) Theoretical growth form of β-hematin viewed along the a- and c-axis, respectively. {hkl} indices of some faces are indicated. Field emission inlens scanning electron microscopy micrographs of hemozoin purified from (c) P. falciparum, (d) S. mansoni, and (e) Hemoproteus columbae. Representative crystals in parts c-e that strongly resemble the theoretical form are delineated.26

to the {01j1} face, which, by comparison, is highly corrugated (Figure 6b). The theoretical morphology (Figure 5a,b) appears to be very similar in both habit and form to that of the hemozoin crystals with distinct faces from various parasites,21 shown delineated in Figure 5c-e. It is noteworthy that the side faces of several specimen crystals of hemozoin from the mammalian Plasmodium species, reported by Noland et al.27 as bricklike with smooth sides at (near) right angles, clearly correspond to the {100} and {010} side faces, the dihedral angle between which is 82°, as is evident from Figure 5b. The possibility that the hemozoin side faces are of the type {110} and {11j0}, the angle between which is 79°, may be ruled out, as discussed above. Scanning electron micrographs (SEM) of bundles of β-hematin crystals reported by Bohle and co-workers28 (Figure 7a) appear to be consistent with the theoretical growth form insofar that the crystals are elongated and terminated by a slanting face. The assigned {hkl} indices of the crystal faces of β-hematin were experimentally established by transmission electron microscopy (TEM) images coupled with electron diffraction (ED) patterns (Figure 7b-d), as reported by Solomonov et al.29 It is interesting that the appearance of well-formed hemozoin and β-hematin displaying slanted end faces with the same angular geometry is a clear indication that the crystals cannot be composed of polymeric material; the latter, which is polydisperse, would have yielded crystals with end faces, not necessarily smooth, but making a right angle with the needle axis.

Armed with knowledge of the crystal morphology of the synthetic and biogenic forms of hemozoin, it became possible to address the role played by quinolines and other antimalarials for inhibiting their crystal nucleation and growth. First is an introduction of a general nature on the inhibition of nucleation and growth of molecular crystals via additives. A salient lesson to be learnt from experiments in this field is that the inhibitor, to be most effective, should bind to the relatively fast-growing crystal faces.30 For example, the antifreeze proteins in fish that inhabit the polar seas act in such a way; they bind to the fastest-growing faces of the ice nuclei to effectively lower the freezing point.31-33 We may classify such inhibitors, which have a structural complementarity to the surface that they bind to but no structural similarity to the molecules of the host crystal, as “capping” molecules (CMOLs). There is another class of crystal nucleation and growth inhibitors, labeled “tailor-made” additives, the molecular structure of which mimics the host molecule present in the solution but for a modified moiety. It is now well-established that minor amounts of such tailor-made additives (TMAs) may induce dramatic changes in the nucleation properties, growth rate, and morphology of the crystal.34,35 This effect occurs via selective adsorption of the additive molecule on those surface sites where the modified moiety emerges from the crystal surface, followed by inhibition of regular deposition of oncoming crystal layers. The adsorption of these two types of auxiliary molecules is schematically depicted in Figure 8. The (001) and (001j) faces of the crystal (Figure 8a) expose inversion-related “little men” (green vs yellow) mimicking the β-hematin cyclic dimer. Thus, the TMA, which is the “little red man” with the black hat, can be stereo- and enantioselectively adsorbed only at green and yellow sites on the (001) and (001j) faces, respectively, as shown in Figure 8b. As for crystal surface adsorption by capping molecules (CMOLs), once again, they can be stereo- and enantioselectively adsorbed only at yellow and green sites on the (001) and (001j) faces, respectively. This adsorption of the auxiliary molecules will hinder the regular deposition of oncoming molecular layers on the (001) and (001j) faces, resulting in growth inhibition, as shown in Figure 8. We shall now examine possible modes of action of antimalarial drugs as CMOLs or TMAs for the inhibition of nucleation/growth of synthetic and natural hemozoin.

3.2. Mode of Action of Antimalarial Quinoline Drugs Quinine (Chart 1, 1) is found in the bark of the Cinchona tree, indigenous to the Andes mountains. The bark’s use as a treatment for malaria was described in the mid 17th century,36 even though the active alkaloid pharmaceutical ingredient, quinine, was not isolated from the bark by Pelletier and Caventou until about 200 years later in 1820. In the 20th century, several synthetic quinoline type drugs such as the aryl-amino-alcohol mefloquine (2) and the 4-aminoquiolines chloroquine (3), amodiaquine (4), and primaquine (7a) came onto the market. Of these, chloroquine became the drug of choice, because of excellent clinical efficacy, low toxicity, and cheapness of manufacture. However, possibly as a result

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Figure 6. Repeating unit cell crystal structure of β-hematin delineated by various low-index {hkl} crystal faces, viewed along (a) the c-axis and (b) the a-axis.

Figure 7. (a) SEM of β-hematin crystals reported by Bohle et al. [Reprinted with permission from ref 28 (http://journals.iucr.org). Copyright 2002 IUCr]. (b-d) TEM images (top) and corresponding selected-area ED patterns (bottom) of β-hematin crystals obtained from (b,c) MeOH-DMSO and (d) CHCl3 solutions. The crystal faces are (hkl) indexed, based on the corresponding ED pattern. The 0kl diffraction pattern shown in part b is from the labeled crystal. The morphologies of three other similar-shaped crystals are shown.29

of widespread use, parasitic-resistant strains to chloroquine, and also other synthetic quinolines, have developed.7,37 Indeed, it is this resistance, made manifest by reduced drug accumulation in the food vacuole,1,7,37,38 that has motivated the need for a fuller understanding of the mode of action of quinoline drugs knowledge that could be put to use for the design of new antimalarials. As already mentioned in section 2.1, Goldberg and coworkers proposed a common mechanism of blockade of hemozoin formation by antimalarial quinolines,15 depicted schematically in Figure 9, when it was still believed that hemozoin was composed of heme polymers. The idea was that a heme-quinoline complex binds to a growing polymer, terminating its growth. According to Moreau el al.39 and O’Neill et al.,40 the principal bonding interactions between the free heme and the drugs chloroquine or amodiaquine comprise π-π stacking forces of the quinoline ring over the porphyrin with possible weak electrostatic interactions

between the protonated ammonium group of the quinoline and the carboxylate group of the heme.

3.2.1. Proposed Binding Sites of Quinoline Antimalarials on Hemozoin Crystal Faces Making use of the theoretical growth form of β-hematin (Figure 5a,b), Buller et al. were able to propose possible binding sites of the antimalarial quinolines on β-hematin’s crystal surfaces.26 One site is the weakly developed (001) and (001j) faces that form at the ends of the β-hematin needle. These two symmetry-related faces are fast growing, which is due to the pronounced {001} crystal surface corrugation and the O-H · · · O hydrogen bond between the propionic acid groups of neighboring molecules along the a-c-direction. The grooves within the corrugated {001} surface (Figure 10a) run parallel to the a-axis, exposing flexible propionic acid

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Figure 8. Schematic representation of β-hematin crystal structure with the cyclic dimer depicted by green and yellow “little men”. (a) The pure crystal arrangement and its morphology. (b) The crystal arrangement and its morphology when grown in the presence of either TMAs or CMOLs.

CH2CH2CO2H moieties, Fe-bound carboxylate O atoms (Fe-O-CdO), vinyl and methyl groups, as well as aromatic surfaces. The quinolines, 1-4 (Chart 1), all protonated at the nonprimary exocyclic amine, can each stereochemically cap ontothe{001}surface26 viaa(quinoline)amine · · · acid(porphyrin) salt bridge and still fit snugly on that surface by intercalation of the quinoline rings between the aromatic groups of the β-hematin molecules, shown for chloroquine in Figure 11a,b and for amodiaquine, quinine, and mefloquine in Figure 12a-c. Chloroquine, and also amodiaquine, form a CCl · · · H3C interaction and an (aromatic)N · · · HC)C(vinyl) hydrogen bond, albeit weak, with the heme surface molecule to help anchor the guest within the crevice. The combination of the acid-base salt bridge and the interactions involving the quinoline ring is achieved by virtue of the six-membered exoquinoline -NCCCCN(CH2CH3)2 chain, of length 6 Å that extends from the R-substituent of the quinoline C4 atom to the flexible amine (assuming an all-trans conformation). The exoquinoline chains of quinine and mefloquine are not as extended and flexible as that of chloroquine. Nevertheless, their antimalarial activity can be understood in terms of binding at the {001} face, via contacts already alluded to, as well as other interactions, shown in Figure 12. These include the (methoxy)O · · · H-O-H · · · O(dC-O-Fe) hydrogen bond between quinine and β-hematin via interleaving water, and the electrostatic CF3 · · · H3C and CF3 · · · HCdC hemozoin-mefloquine contacts). Water is also proposed to act as a glue linking the p-OH substituent of amodiaquine to the carbonyl O atom of an exposed OdC-O-Fe moiety of β-hematin, according to Figure 12a. A quinoline such as chloroquine may bind to the {100} face via the (quinoline) amine · · · (β-hematin) propionic acid salt bridge, the acid moiety exposed at the crystal surface being sufficiently flexible to adopt an appropriate conformation, as shown in Figure 13a,b. By taking advantage of the length of the exocyclic chain, the quinoline ring of chloroquine may also form a “T” type contact with the methyl and vinyl C-H substituents of the heme rings. Such a motif is prevalent in herringbone arrangements of aromatics, documented by Desiraju and Steiner.41 The above model leads us to consider the molecular action of antimalarial drugs somewhat similar in structure to the quinolines but which incorporate larger aromatic moieties

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or added groups in the exocyclic chain, such as mepacrine 5, the phenanthrene derivatives halofantrine 8 and lumefantrine 9, and the “reversed chloroquine” 6. Simple packing considerations suggest that mepacrine, which was used extensively in World War II in the Pacific as an effective drug against the blood stages of P. falciparum, can be intercalated at the {001} face in a manner akin to quinolines 3 and 4 but not the phenanthrene derivatives 8 and 9. Nevertheless, this impasse may be circumvented, since 8 and perhaps 9 can make use of their exocyclic moieties (HOCCCN and HOCCN, respectively) to form both the acid-base (NH+ · · · -O2C) bridge and also an O-H · · · Od C-O(-Fe) hydrogen bond at the {001} face and as well make “T” type contacts with the {001} surface as shown in Figure 14a. Alternatively, the phenanthrenes can make similar contacts at the {011} face, which is relatively flat (Figure 9b). We may also envisage that these phenanthrene derivatives 8 and 9 can be bound at the {100} face in a manner akin to that of chloroquine (Figure 13) but perhaps more effectively by virtue of their larger aromatic areas. In view of a recently reported crystal structure by Egan et al.42 of a complex between halofantrine 8 and Fe3+-PPIX, we envisage that such a complex may efficiently bind, as a TMA, to the {010} face of hemozoin and thus inhibit growth along the b-direction, as depicted in Figure 14b,c. Thus, we propose that halofantrine, by involving its free and Fe3+-PPIX complex forms, inhibits hemozoin growth along all three principal crystal axes. Solution studies42 of quinolinemethanols (quinine and quinidene) complexes with Fe3+PPIX suggest that these drugs would act in a manner akin to 8. The antimalarial 6 belongs to a class of recently developed hybrid molecules reported by Peyton and co-workers,43 termed “reversed chloroquines”. The drug is designed to incorporate a reversal agent portion (depicted within the rectangle in 6) that is known to inhibit the export of chloroquine out of the digestive vacuole of chloroquineresistant strains of P. falciparum. The drug was found, by in vitro studies, to be about 10 times more effective against chloroquine-resistant P. falciparum than chloroquine by itself and about twice as effective against a nonresistant strain of the parasite. The reason therefore may also be due to a stronger binding of the hybrid 6 to the {001} and {100} crystal surfaces of hemozoin than chloroquine itself. This would be a result, in part, of an N-H · · · OdC-O(-Fe) bond between the tertiary amine of the reversal agent portion and the free O atom of the Fe-bound carboxylate, assuming that the N atom is protonated in the acidic digestive vacuole. Peyton and co-workers regard the prototype 6 as probably too hydrophobic for human use, although they demonstrated some oral bioavailability in mice.

3.2.2. Experimental Evidence of Quinoline Binding to Hemozoin and β-Hematin Crystal Surfaces Evidence of quinoline binding to the crystal surfaces of hemozoin was first reported by Goldberg and co-workers in 1996.15 They examined cultured parasites incubated with subinhibitory doses of {3H}chloroquine. Making use of electron microscope autoradiography, they found {3H}chloroquine located over the crystals of hemozoin; indeed, the majority of {3H} signal is directly over the crystals, as is evident by the three clusters of dark crystals in Figure 15. The dark crystals most likely expose the {100} or {010} needle side faces. Assuming the former possibility

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Chart 1. Several Antimalarial Drugs of Current or Past Importance, Comprising an Aromatic Quinoline or Phenanthrene Ring and an Exocyclic Chain Moiety Incorporating a Tertiary Amine and Other Groups Possibly Capable of Forming Hydrogen Bondsa

a

Note that the squiggly lines in 3, 5, and 7-9 indicate the presence of a chiral substituent (either CH3 or OH) in racemic form.

Figure 9. Model of quinoline inhibition of hemozoin formation proposed in 1996 by Goldberg et al. when it was still believed that hemozoin was a polymeric material. Reprinted with permission from ref 15. Copyright 1996 National Academy of Sciences U.S.A.

suggests stereoselective adsorption of {3H}chloroquine to the {100} face, as depicted in Figure 13, involving the acid-base bridge, and the “T” type interaction between the C-H groups of the porphyrin rings and the aromatic face of the quinoline ring. We tend to discard the possibility that these dark faces are of the type {010} for that would mean binding of chloroquine to the {010} face involving a poor overlap between the quinoline ring and the exposed {010} surface of the porphyrin ring, as appears evident from the nature of the {010} surface structure of β-hematin (Figure 6b). It is also not inconceivable that the {3H}chloroquine was adsorbed and then occluded, during growth, into the crystal bulk via the {001} or {011} end faces, making the whole crystal look dark. The lighter contrast of the nine parallel crystals inscribed within the white polygon in Figure 15 and other such crystals of lighter contrast might be explained assuming the crystals nucleated at a later stage. The darker areas at the {001} or {011} end faces terminating the longer sides of several of these crystals suggest drug

accumulation thereon, in accordance with the theoretical model, as proposed by Buller et al.26 The weakness of the above interpretation, reinforced by doubts thereon raised by Egan in a review on hemozoin as a unique crystalline drug target,11 prompted the need for unambiguous evidence, indicating stereoselective adsorption of quinoline drugs to identifiable faces of β-hematin (or hemozoin) and whether these faces are consistent with the surface-binding models described above. This aim was addressed as follows. β-Hematin crystals grown from DMSO-MeOH or CHCl3 solutions in the presence of chloroquine and of quinine additives displayed, in a significant amount, crystals tapered at both ends, which are highly symmetric in shape (Figure 16a).29 We proposed that such tapering was adopted to help reduce inhibition of growth along the c-axis by adsorption of the quinoline additive on the {001} or {011} facets expressed as ledges in the steppedface morphology shown in Figure 16b, resulting in a spine formation of thinner and thinner cross-sections. Analysis of synchrotron PXRD data of pure and affected β-hematin, grown in the absence and the presence of quinine and chloroquine, respectively, suggested for the crystals grown in chloroform solution that the additives reduce the crystal mosaic domain size along the c-direction, which is consistent with the crystal growth experiments. Coherent grazing X-ray diffraction data of pure and affected β-hematin, the latter obtained from MeOH:DMSO solution, were consistent with the above conclusion; the pure crystals yielded a strong {100} speckled diffraction peak, and the affected crystals gave only very weak diffractions signals that disappeared with time. Vibrational micro-Raman and attenuated total reflectance infrared spectroscopy showed no direct evidence of the

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Figure 10. Crystal structure of β-hematin, viewed along the a-axis, showing the molecules exposed at (a) the corrugated {001} face, with crevices outlined in green, and (b) the relatively flat {011} face.

Figure 11. Chloroquine bound to the (001) face of a β-hematin crystal highlighting energetically favorable interactions. The structure is viewed (a) along a general direction and (b) the crevice along the a-axis within which the drug is intercalated. (c) A schematic view depicting the acid-base, C-H · · · N and C-H3 · · · Cl interactions. The inset is an experimental charge-density distribution of parts of an aromatic molecule arranged to form a hypothetical C-H · · · N hydrogen bond, highlighting its electrostatic nature.

Figure 12. Favorable polar contacts (in Å) between β-hematin at the {001} crystal surface and intercalated (a) amodiaquine, (b) quinine, and (c) mefloquine. The inset shows the experimentally derived charge density distribution of a CF2 group, with reference to the charge distribution of the CF3 groups of mefloquine.

presence of quinine or chloroquine but did display minor but distinct changes between quinoline-affected and pure

β-hematin crystals.29 These changes were explained in terms of the effect of stereoselectively bound quinoline occluded

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Figure 13. Intermolecular contacts between chloroquine and β-hematin at the {100} crystal surface. Views: (a) edge-on to the plane of the (100) face and (b) perpendicular to the plane of the (100) face. (c) Experimentally derived deformation electron density distribution of a phenyl ring.

within the bulk of the crystal and adsorbed on the crystal surface. All in all, the experimental evidence seems to be consistent with the model of quinoline binding to the {001}, {011}, and {100} faces of hemozoin, leading to inhibition of crystal nucleation and growth. During this process, a quinoline molecule may also bind to a β-hematin molecular dimer before the latter is adsorbed on a crystal surface, which has a kinetic disadvantage that the complex must “locate” the appropriate face, say (001) or (001j) on the opposite side of the crystal, onto which to adsorb, depending upon to which of the two carboxyl groups of a β-hematin molecular dimer the drug is bound. Naturally, if both carboxyl groups are bound to quinoline, such a complex cannot be stereoselectively adsorbed onto the crystal surface and so will be ineffective as an inhibitor. Here, we note evidence, albeit indirect, in favor of the presence of the β-hematin molecular dimer in solution according to UV-vis absorption spectroscopy measurements monitoring the formation of β-hematin by gradually acidifying a solution of hemin dissolved in a solution of 0.1 M NaOH.44

3.3. Hemozoin as a Target of Antimalarials Containing Two Amino-Terminating Chains Now, we review the possibility of hemozoin nucleation/ growth inhibition as an important factor in the antimalarial activity of drugs other than the quinolines and phenanthrenes, focusing primarily on diethylamino-alkoxyxanthones, a molecule that contains a central rigid xanthone moiety flanked by two flexible aliphatic chains of variable length, each terminated by a tertiary amino group.

3.3.1. The 3,6-Bis-ω-diethylamino-alkoxyxanthone Series The 3,6-bis-ω-diethylamino-alkoxyxanthones (Chart 2, 10) belong to a novel class of antimalarial compounds with activity against multidrug-resistant Plasmodium parasites, reported by Riscoe and co-workers.45,46 These compounds, labeled XNn, where n is the number of CH2 groups per chain ranging from 2 to 8 (except n ) 7), form soluble complexes with heme and prevent the precipitation of β-hematin in aqueous solution under the mildly acidic conditions of the parasitic digestive vacuole at pH ) 4.8 ( 0.4. A strong correlation was found between the heme affinity measured in aqueous solution, the alkoxy chain length, and the antimalarial potency.45 The antimalarial activity of the drugs was explained by Riscoe and co-workers45,46 in terms of the binding constant of the drug to heme in solution.

As an additional, or alternative, possibility, we proposed hemozoin nuclei as a primary drug target, involving binding of the drug to the {001} and perhaps {100} crystal faces, leading to inhibition of crystal nucleation/growth.29 The drug XN5 has chain lengths appropriate for its two protonated amino groups to bind simultaneously to two charged carboxyl groups, separated by a lattice distance of 2a and exposed at the {001} faces, and also form favorable van der Waals contacts with the crystal face (Figure 17). By comparison, while one protonated amino group of XN2 may bind to a charged carboxyl group, the other protonated amino group would form a hydrogen bond with O(dC-O-Fe), certainly a weaker interaction. This crystal surface binding model also accounts for the reduced antimalarial activity for molecule XN8 being about 6 Å longer than XN5 and so would bind to the two surface carboxyl groups on the {001} or {011} faces only if the molecular chain would be bent and thus make poorer van der Waals contact with the crystal surface. Binding to the {100} face is also possible since the intramolecular distance of ∼24 Å between the two protonated amino groups of XN5 matches the lattice spacing 3c of 24 Å between propionic acid groups. This analysis is also in agreement with the suggestion by Buller et al. on the use of antimalarial drugs that would bind to two or more carboxylic acid surface sites on the {001} face.26 The efficient antimalarial potency of diamidines, another class of novel drugs embodying proton donor groups (amidine) at either end of the molecule, has been reported some years ago.47 These authors showed that diamidines inhibit hemozoin formation in vitro with a similar potency to chloroquine. It is likely that these drugs, such as pentamidine (H3N2C-C6H4-O-C5H10-O-C6H4-CN2H3) inhibit hemozoin formation as a result of binding of both amidine groups of the molecule to exposed propionic acid groups at the crystal surface. A primary difference between the diethylamino-alkoxyxanthones and the diamidines is that while the latter contains a large and rigid central aromatic moiety flanked by two flexible alkylamino chains, the former incorporates a flexible central alkyl chain flanked by two phenyl-amidine groups.

3.4. The Arteminsinin Type Drugs as Tailor-Made Inhibitor of Hemozoin Growth Finally, we consider inhibition of crystal nucleation/growth of hemozoin as a possible mode of action to account for the widely used antimalarial drug, artemisinin, extracted from an ancient Chinese herbal remedy, Artemisia annua (sweet wormwood or “qinghao”). Artemisinin 11a (Chart 3a) is a

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Figure 15. Electron microscope autoradiograph in the digestive vacuole of a cultured parasite of P. falciparum, incubated with subinhibitory doses of {3H}chloroquine reported by Goldberg et al. The three dark clusters represent malaria pigment crystals covered by {3H}chloroquine. The array of parallel crystals within the white polygon is less dark except for the regions at the crystal ends indicated by representative arrows, suggesting excess location of {3H}chloroquine at these {001} end faces. Reprinted with permission from ref 15. Copyright 1996 National Academy of Sciences U.S.A.

Figure 14. (a) Intermolecular contacts between halofantrine 8 and β-hematin at the {001} crystal surface viewed perpendicular to the plane of the face. (b,c) Binding of the complex of halofantrine with Fe3+-PPIX (formed via coordination between the OH group of 8 and Fe3+ as well as a N-H+ · · · -O2C salt bridge) to the {010} face, viewed perpendicular and parallel to the face, respectively. Note that the free acid group of Fe3+-PPIX in the complex participates in a regular O-H · · · O bond with a neighboring host heme.

1,2,4-trioxane that has been used in China for the treatment of multidrug resistant P. falciparum malaria, but its low solubility in both oil and water has led to a search for more effective and water-soluble drugs such as 11b and 11c. Unlike the other antimalarial drugs discussed above, which may be regarded as hemozoin surface capping agents, artemisinin as an adduct of a β-hematin molecular dimer may be envisaged as a TMA, whose properties are described in

Figure 16. (a) Symmetrically tapered crystals of β-hematin obtained from growth in the presence of the quinoline drugs. (b) Model of stepped face morphology showing {001} ledges to account for the symmetric tapering.29

section 3.1 and depicted in Figure 8b, in the way it may be adsorbed on the hemozoin crystal surface. Artemisinin and its derivatives (11a-c), being highly active against multidrug-resistant strains of malaria parasite, have attracted much attention; its mechanisms of action, resistance, and toxicity have been recently reviewed by Meshnick48 and by Bray et al.7 The reactivity of the peroxide function of artemisinin has been considered as the primary factor of the pharmacological activity. A key finding is that artemisinin cannot be cyclically oxidized and reduced; only

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Chart 2. Generalized Structure of 3,6-Bis-ω-N,N-diethylamino-alkoxyxanthones, Labeled XNn, Where n Is the Number of CH2 Groups

one free radical can result from one drug molecule. The selective toxicity of artemisinin may arise from alkylation of the heme, leading to heme-artemisinin-derived covalent adducts. Heme-artemisinin adducts have been demonstrated in parasite cultures treated with therapeutic concentrations of artemisinin derivatives.49 Artemisinin forms covalent bonds with heme when incubated in a cell-free solution, and these same artemisinin-heme adducts appear to form in artemisinin-treated parasites. At micromolar concentrations, artemisinin inhibits hemoglobin digestion by malaria parasites and inhibits hemozoin formation, but this has only been demonstrated in cell-free conditions.50 The structure of an artemisinin-porphyrin adduct has been characterized by Meunier and co-workers,51 following activation of the peroxide function of artemisinin by iron (II)-heme generated in situ from Fe(III)-protoporphyrin-IX and glutathione, a biologically relevant reductant. Such a reaction produced a high yield (85%) of heme derivatives alkylated at R-, β-, γ-, and δ-positions by a C4-centered radical derived from artemisinin (12), where the regioselectivity at the R-, β-, and δ-positions were about the same at 28% on average. We made use of this structural information to develop a scenario involving hemozoin nucleation inhibition to account for the antimalarial behavior of artemisinin and, as an extension, rationalize the increased antimalarial efficacy when artemisinin and quinoline type drugs are used in conjunction.29 However, there are various hypotheses on the mechanism of artemisinin-bases drug action; besides heme alkylation, a number of other biological targets have been proposed including specific parasite proteins.7,48 We described a model involving reaction of four artemisinin-heme type adducts (12), with a free heme monomer to yield eight derivatives of the cyclic β-hematin dimer, comprising four diasteroisomeric pairs, which may bind to the {100}, {010}, and {001} faces of a β-hematin crystal, shown in Figure 18.29 Such molecules, if present in the digestive vacuole of the parasite, would act in a manner akin to a classic TMA in solution designed to inhibit crystal nucleation and growth.30 According to this model, crystal growth of hemozoin may be inhibited along all three principal crystal directions a, b, and c. We stress that efficient inhibition of hemozoin formation via artemisinin would seem possible provided the cyclic β-hematin dimer derivatives are formed in the digestive vacuole of the parasite; the artemisinin-heme type adducts (12) would not be as strongly adsorbed on hemozoin crystal surfaces to act as efficient growth inhibitors.

3.5. Rationale for Antimalarial Drug Design with Hemozoin Crystal Surfaces as a Target The challenge in the design of antimalarial drugs active in the RBC stage is to take advantage of the different functional groups on the different crystal surfaces to act as receptors to the drug so that it may be adsorbed simulta-

Figure 17. Packing arrangement of β-hematin illustrating the binding to the (001) face of diethylamino-hydroxyxanthone molecules XN2 shown in parts a and b and XN5 shown in parts c and d. Views parallel to the (001) plane (dashed yellow line) are depicted in parts a and c, and views perpendicular to the (001) plane are shown in parts b and d. The C and H atoms of hydroxyxanthone are colored in orange, the N atoms are in blue, and the O atoms are in red. The carboxylate O atoms of β-hematin in contact with the NH groups of hydroxyxanthone are large red spheres.29 Chart 3. (a) Molecular Structure of Artemisinin and Two Derivatives and (b) Alkylation of Iron(II)-Heme by Artemisinina

a In these heme-artemisinin adducts, the substitution may occur at R-, β- (as shown), γ-, or δ-positions, yielding four different isomeric products ARH, AβH, AγH, and AδH, where A represents an artemisinin moiety covalently bound to a heme H via sites R, β, γ, or δ.

neously onto different crystal faces of hemozoin and thus inhibit crystal nucleation/growth more effectively. With this aim in mind, we now focus on the principles involved in the design of surface CMOLs and TMAs to inhibit growth of hemozoin.

3.5.1. Design of Hemozoin Surface CMOLs for Growth Inhibition The hemozoin crystal receptor sites capable of forming strong interactions with a capping additive molecule are the propionic acid and carboxylate-iron OdC-O(-Fe) moieties, various C-H groups on the periphery of the porphyrin ring, and the surface of the latter. All of these sites appear on the end faces {001}, {01j1}, and {011} (see Figure 6) of crystals

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j BH, AδBH, and Aδ j BH, where Figure 18. Arrangement of six different artemisinin-β-hematin dimer adducts (ARBH, AR j BH, AβBH, Aβ A represents an artemisinin moiety covalently bound to a heme H, R, β, and δ are the three peripheral sites on the heme shown in Chart 3b, and BH is the molecular dimer) on crystal faces of β-hematin. (a) General view of adsorption of the adducts, to the four {100} and {010} faces, seen along the c-axis. (b,c) Detailed views along the c- and a-axes of adsorption of the adducts on the various faces, where the molecular structure of the artemisinin moiety (A) bound to the hematin dimer at the different peripheral sites had been modeled by energy minimization.29

of hemozoin extended along the c-axis. The {001} and {01j1} faces are highly corrugated, permitting intercalation of planar aromatic quinoline rings between neighboring porphyrin rings, but the faces are fast-growing and therefore morphologically not well-expressed. By comparison, the {011} face, being relatively flat, has the disadvantage that an adsorbate quinoline ring cannot be intercalated between neighboring porphyrin rings. On the other hand, the face is well-expressed so that the phenanthrene antimalarials 8 and 9, whose aromatic moieties are too large to be well-intercalated at the {001} or {01j1} faces, can be easily bound to the {011} face and still be linked to the CO2H and OdC-O(-Fe) moieties. The flat {100} and {010} side faces are most well-expressed in hemozoin needle-shaped crystals. The {100} face exposes the flexible propionic acid moiety, although at an oblique angle, yet whose orientation can be easily adjusted. The face also exposes peripheral heme C-H groups. The {010} face exposes only the surface of heme rings at an angle not particularly suitable for plane-to-plane contact with an aromatic system (e.g., quinoline or phenanthrene). The {(110} faces (Figure 6) expose CO2H, C-H groups and porphyrin surface receptor sites and so should be suitable for drug binding, but the faces have not been observed, presumably because they are highly corrugated. Drug functional groups capable of forming a strong link to the propionic acid crystal surface receptor site via H-bonds are primary amides (CONH2) and amines, the latter via an acid-base NH+ · · · -O2C interaction. The primary amides can form a cyclic H-bonded heterodimer with the propionic acid moiety,52 but the amine is preferred; it has been found that the protonated state of the exocylic tertiary amine of antimalarials such as amodiaquine and chloroquine assists in retarding the drug diffusion from the acidic vacuole, thus reducingcriticaldrugamountforcrystalgrowthinhibition.19,53,54 Regarding the carboxylate OdC-O(-Fe) receptor site, it can act as an acceptor to proton donors, such as O-H and

N-H, which is consistent with a search of the Cambridge Structural Database of OH and NH groups H-bonded to the free O atom of a carboxylate group bound to pentacoordinated Fe atom that yielded 14 positive hits. A measure of the proton acceptor strength of the OdC-O(-Fe) receptor site was gleaned via density functional theory (DFT) computations of methanol H-bonded to acetic acid (HO2CCH3) and to OdC(-CH3)-O-Fe, in which the Fe atom is part of a Fe(III)-protoporphyrin IX ring.55 The computation shows that interaction with the Fe(III)-protoporphyrin IX center significantly influences the geometry and electronic structure of the carboxyl group (see Figure 19); the two C-O bonds become more equal in length, there is an increase in the charge density of the free O atom, as is manifest in the electrostatic potential surfaces of the two molecules, and there is a significant increase in the Hbonding binding energy (BE) of methanol to the OdC-O(-Fe) atom. The C-H proton donor groups available on the periphery of the porphyrin ring may be either a vinyl (CdC-H) or a methyl (CH3). The CdC-H group may act as a donor to N(aromatic) or O(carbonyl) lone pair electrons or to an electronegative F-substituent, of the drug. The existence of C-H · · · N(aromatic) interactions in crystalline solids and their role in determining molecular packing is now wellestablished.41,56 The electrostatic nature of this interaction is depicted in Figure 11c inset, which shows an experimentally derived charge density distribution of an aromatic molecule57 involved in a hypothetical C-H · · · N bond. The crucial role played by the C-H · · · N(aromatic) becomes clear when comparing the antimalarial behavior of chloroquine 3, primaquine 7a, and pamaquine 7b. The latter two each incorporate extended exocyclic amine moieties, akin to chloroquine, however, at the C8 position, precluding formation of the (hemozoin)CH · · · N(drug) 3.4 Å H-bond that would be replaced by (hemozoin)CH · · · HC(drug) contact of

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Figure 19. Comparison, based on DFT computations, of electronic structure of acetic groups in (a,b,e) pure acetic acid and (c,d,f) Fe(III)-protoporphyrin IX. (a,c) Natural atomic charges (from natural bond orbitals), (b,d) optimized geometries and corresponding binding energies of H-bonded complexes with methanol, and (e,f) electrostatic potential contour plots (contour interval ) 0.004 au).

∼4 Å. In addition, the (hemozoin)CH3 · · · Cl(drug) interaction is also absent. Thus, it is not completely surprising that both 7a and 7b are inactive for the RBC stage of malaria, being poor inhibitors of hemozoin growth,19 although 7b is effective in the liver stages of P. ViVax infections.58 The CH3 group may form an interaction with an electronegative Cl or F3 substituent, as has been briefly mentioned in section 3.2.1, involving binding of chloroquine and mefloquine, respectively, to the hemozoin crystal surface. It is noteworthy that the (C)H atoms have a net positive charge of about 0.06 electron units, and the F atom has a net negative charge of 0.14 e.u., according to experimental deformation electron density distribution in a CF2 system,59 shown in the inset of Figure 12c, consistent with the model and report of the existence of C-H · · · F hydrogen bonds.41 The CdC-H or CH3 groups may also form on the {100} face of hemozoin a “T” type interaction with the charge density of an aromatic moiety of the drug such as the quinoline or a phenanthrene ring. As already mentioned in section 3.2.1, such a motif is prevalent in herringbone arrangements of aromatics;41 it involves interatomic dispersion forces and electrostatic interactions between the polar C-H bonds and the net electron charges in the bonds. Such a charge distribution in an aromatic ring (Figure 14c) was derived from a deformation electron density distribution study,60 yielding an integrated charge of about 0.2 e.u.61 Finally, the porphyrin-exposed crevice along the a-axis on the {001} or {01j1} crystal surfaces may be used for the intercalation of drug aromatic rings therewithin. Other crystal surfaces such as the {010}, possibly {110}, and {11j0} also expose porphyrin rings, but the porphyrin-drug ring interface contact occurs only on one side of the drug. The importance of taking advantage of the relative spatial arrangement of the various receptor groups on the different hemozoin crystal faces in the design of antimalarial drugs is clearly revealed by the proposed interactions chloroquine makes with the surface receptor groups within a crevice along the a-axis at the corrugated {001} surface (NH+ · · · O2-C, -Cl · · · H3C-, N · · · HCdC, and plane-to-plane contacts between quinoline and porphyrin rings), as depicted in Figure 11. A crucial element here is the exocyclic amine chain,

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which is sufficiently long and flexible to ensure optimal binding of the distant amine to the host acid moiety, allow appropriate intercalation of the quinoline, and permit the polar interactions. The importance of the exocyclic chain length is also substantiated by a study by Ridley et al.,62 who performed an in vivo study on the effect of different flexible chain length analogues of chloroquine on β-hematin crystal growth inhibition, making use of chloroquine-sensitive parasites. They report that the drug with a 3 Å chain length induced IC50 values,63 higher by a factor of ∼1.5 as compared with chloroquine. Another example pinpointing the importance of the relative arrangement of the surface receptor groups is the proposed interaction at the {001} or {011} faces involving adsorbate exocyclic chain moieties, which embody substituents that can be simultaneously bound to the hemozoin propionic acid and carboxylate OdC-O(-Fe) groups, which are almost parallel to each other and separated, intramolecularly along the a-axis, by ∼5 Å. This distance almost matches the separation between the tertiary amine and the hydroxyl group in halofantrine 8 and lumefantrine 9, but to a lesser extent, which is consistent with the fact that 9 is a less effective antimalarial than 8.10 Here, we note that the phenanthrene rings are too large to fit snugly within the crevice on the {001} face (Figure 14) but can easily lie on the {011} face making “T” type contacts therewith. Also worthy of mention is the “reversed chloroquine” 6, with an intramolecular distance of ∼5 Å between tertiary amine moieties along the exoquinoline chain, which we propose binds simultaneously via salt bridges to CH2CH2CO2- and OdC-O(-Fe) on the {001} face, to help account for its excellent antimalarial behavior,43 already mentioned in section 3.2.1. Still focusing on the propionic and OdC-O(-Fe) moieties exposed at the {001} and {011} faces, these two groups repeat by translation along the a-axis of length 12 Å. This set of surface receptor sites may be exploited by the use of a drug with say a pair of protonated amine or amidine groups whose separation matches the distance between the exposed propionic acid moieties. Indeed, the antimalarial behavior of the diethylamino-alkoxyxanthones series 10 was explained by such adsorbate-{001}surface interactions (see section 3.3.1 and Figure 17). Now, we turn to possible use of receptor sites on the {100} face, which exposes a two-dimensional array of propionic acid groups (of dimension 14.7 × 8.0 Å2), as well as C-H bonds. The experimental evidence presented for binding of a quinoline drug, such as chloroquine, to the {100} face is not caste in iron (see section 3.2.2 and Figure 15); yet, according to the model (Figure 13), the chloroquine makes, besides an NH+ · · · O2-C salt bridge, the “T” type face-onedge contact between the quinoline ring and the peripheral porphyrin C-H groups, taking advantage of the size of the quinoline ring and the length and flexibility of the exocyclic chain. It is not inconceivable that the two tertiary amine groups of “reversed chloroquine”, separated by ∼5 Å, bind to two propionic groups separated by the translation c-axis of 8 Å, assuming that they are sufficiently flexible to span the difference of 3 Å. The diethylamino-alkoxyxanthones 10 should also be able to bind for the molecule XN5, n ) 5 (see Chart 2), via its two amine groups, to the {100} face, given that the intramolecular separation of 24 Å between the two amino groups matches the lattice distance 3c, as discussed in section 3.3.1. The drug should also form the “T” type contact between its xanthone ring and the exposed

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porphyrin C-H groups of hemozoin. It is also perhaps possible that the phenanthrenes 8 and 9, which cannot intercalate efficiently within the crevice at the {001} face, can make use of the larger cross-sectional area of the phenanthrene ring to allow tight binding to the {100} face of hemozoin via the “T” type contacts. The antimalarial quinolines are either nonchiral, as amodiaquine 4, or chiral, the latter either racemic, as 2, 3, 5, or 7-9, or homochiral, as the naturally occurring quinine 1, which contains four chiral C centers. The role played by the molecular chirality for inhibiting hemozoin crystal growth may be appreciated given that the crystal structure is centrosymmetric P1j, so that the opposite (hkl) and (-h-k-l) faces of hemozoin are chiral but of opposite handedness, namely, enantiotopic, as are the (001) and (001j) faces in Figure 8a. The binding of chiral CMOLs or TMAs to chiral crystal surfaces has already been outlined in section 3.1 and Figure 8b. Thus, in principle, the binding energies of a chiral quinoline of one handedness, with a particular (hkl) face of hemozoin, will be the same as that of the chiral molecule of opposite handedness with the (-h-k-l) face. Obviously, the binding energies of a chiral quinoline of one handedness with (hkl) and (-h-k-l) faces of hemozoin must be different. Therefore, the best strategy for the design of crystal growth inhibitors of hemozoin in the digestive vacuole of the parasite would be to make use of nonchiral quinoline drugs because they bind equally well to either (hkl) or (-h-k-l) faces, followed by racemic quinolines, since each of the two enantiomers may have to “locate” the appropriate chiral face. In this section, we discussed the design of antimalarial CMOLs capable of retarding growth along two principal growth directions a and c. However, growth may still occur, albeit slowly, along the b-direction at the {010} face. It is noteworthy that halofantrine 8, as well as the quinolinemethanols 1 and 2, might form complexes with Fe3+-PPIX42 to act as TMAs (see section 3.1 and Figure 8b) that could bind to the {010} face (Figure 14b,c), resulting in growth retardation along the third principal direction, b. This proposal leads us to models of TMAs able of inhibiting growth of hemozoin along all of its three principal axes, discussed in the next section.

3.5.2. Hemozoin Growth Inhibitors as TMAs that Resemble the β-Hematin Molecular Dimer Here, the focus is on another class of hemozoin growth inhibitors, the molecular structure of which resembles the β-hematin cyclic dimer, but for an added moiety. The TMA appropriate for inhibition of hemozoin growth is the β-hematin cyclic dimer preferentially alkylated at each of the peripheral porphyrin positions, R, β, and δ (as shown in Chart 3b) of one of the two porphyrin rings of the dimer molecule. To date, the only reported drugs that may, in principle, form TMAs are the artemisinins, for example, 11a-c. As described in section 3.4, artemisinin can alkylate the heme primarily at the R-, β-, and δ-positions with equal regioselectivity to yield the adduct 12. We then postulated that such three heme adducts form cyclic dimers with pure heme monomers to yield molecular structures akin to that of the β-hematin dimer and, then, in principle, bind to the different crystal faces of hemozoin, as depicted in Figure 18, and inhibit their growth in all directions.29 On the assumption that the mode of antimalarial action of the artemisinins follows the above route, we may predict that the synthetic

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peroxide analogues, whose antimalarial activity has been recently reviewed by Bray et al.,7 might act similarly.

3.5.3. Antimalarial Combination Therapy Involving Artemisinin and Quinoline Type Drugs According to the above-described model, since only a small fraction of the ensemble of artemisinin cyclic β-hematin dimer adducts might be adsorbed onto the small but fast-growing {001} faces, adsorption would occur primarily on the large but slow-growing {100} and {010} faces. The quinoline drugs, on the other hand, are expected to be bound primarily to the fast-growing {001} faces at the opposite ends of the needle c-axis and perhaps on the slow-growing {100} face along the a-axis. Thus, we may rationalize why artemisinin-based combination therapy (ACT) involving artemisinin and quinoline drugs is more effective than each of them applied separately in clinical studies combating malaria.64-66 The quinoline drugs are expected to inhibit growth primarily along the fast-growing needle c-axis and perhaps along the slow-growing a-axis, and the artemisinin type drugs have been hypothesized to retard growth along all three principal crystal directions but mainly along a and b. Thus, a combination of these two types of drugs would be effective inhibitors of overall crystal formation of hemozoin.

4. Nucleation of Hemozoin Crystals Little is yet known at the molecular level on the nucleation process of hemozoin in the digestive vacuole of the parasite, particularly the role played by various molecules to promote the process, and thus is a topic of active current interest. Nevertheless, a discussion of pertinent results over the past two decades might shed some light on possible mechanisms of hemozoin formation. We first focus on a key experiment reported in 1990 by Goldberg and co-workers on hemoglobin degradation in P. falciparum;67 they observed aligned parallelepiped crystals of hemozoin formed in the digestive vacuole of the parasite, displayed in the transmission electron micrograph (Figure 20). The authors were struck by the fact that photographs of intact parasites show pigment crystals lined up along a single axis, whereas photographs of isolated vacuoles repeatedly show a disordered array. They suggested that the alignment may merely be an artifact of isolation or fixation or, considering the paramagnetic properties of the iron in hemozoin, that the pH gradient across the vacuolar membrane establishes an electromotive force across the vacuole that causes the pigment crystals to align within the magnetic field so generated. During isolation, the factors needed to maintain a trans-membrane pH gradient may be lost, and the crystals would become disordered. We proposed an alternative mechanism involving epitaxial nucleation of the hemozoin crystals by the surface of the vacuole membrane, namely, via a lipid layer,29 which is consistent with the observation that not only are the crystals aligned along a single axis, but they appear to expose the same crystal face, namely, appear to be parallel. Three mechanisms of hemozoin crystallization within the digestive vacuole are discussed in the literature: promotion byhistidine-richproteins(HRP)intheaqueousenvironment,68,69 promotion by polar membrane lipids,70 and neutral lipid bodies.71-74 The first mechanism has been contested on the basis of two different studies, one reporting the HRP location

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association with lipids in three unrelated organisms. On the assumption that the nucleation is induced within the lipid body by glycerol groups of neutral lipids, exposing OH, CH, and oxygen lone-pair electrons, we may expect nucleation to occur via the {100} face, which exposes -CO2H and C-H groups.

5. Summary

Figure 20. Electron micrograph of the digestive vacuole (dv) of a P. falciparum trophozoite inside a RBC, showing a cluster of aligned hemozoin crystals. Reprinted with permission from ref 67. Copyright 1990 National Academy of Sciences U.S.A.

to be outside the digestive vacuole and the second demonstrating that P. falciparum clone lacking the genes for both HRP II and HRP III forms hemozoin normally.73 Regarding induced nucleation by membranes, it is now well-established that, in general, crystalline monolayers of amphiphilic molecules induce oriented nucleation of molecular and inorganic crystals by virtue of structural complementarity and/or lattice epitaxy between the head groups of the monolayer and the layer arrangement within the crystal.30 Given that the crystals of synthetic and biogenic hemozoin tend to grow fastest along the c-axis and are delineated by sharp {100} and {010} side faces, the crystals in Figure 20 would appear to be aligned parallel to c, so that the nucleation occurs via either the {100} or {010} faces. We may preclude that nucleation would occur via the {(110} side faces since they are highly corrugated (see Figure 6) and so will not be nucleated by a flat lipid surface. It is not possible, however, to determine by visual inspection which of the crystals in Figure 14 are {100} or {010}, since the end faces are not sufficiently well-formed. Regarding the effect by polar membrane lipids, Trager75 demonstrated robust hemozoin formation in P. falciparum axenic cultures, which lack a parasitophorus vacuole membrane. The role of neutral lipids on hemozoin crystallization has been suggested by Fitch et al.76 Egan and co-workers77 recently reported fast β-hematin formation near octanol/ water, pentanol/water, and lipid solution/water interfaces. The presence of lipid bodies composed of di- and triacylglycerols closely associated with the digestive vacuole has been reported by Jackson et al.,71 who postulated the role of the latter in hemozoin crystallization. Of particular relevance is the study by Sullivan and co-workers74 who found neutral lipid nanospheres that envelop hemozoin crystals inside P. falciparum digestive vacuoles. These authors have also identified monoacyl- and diacyl-glycerol lipids in close association with the purified hemozoin. Furthermore, a recent review by Egan73 highlights incontrovertible evidence that hemozoin formation occurs in

We review the interplay between malaria, the formation of synthetic β-hematin and natural crystalline hemozoin, and antimalarial drug action and design. The crystal morphology of β-hematin and natural hemozoin is described, including a simple analysis of why only very few types of stable crystal faces may be expected in keeping with observation. Drug action of various types of antimalarials, such as quinolines, phenanthrene derivatives, and diethylaminoalkoxyxanthones is postulated as a result of stereospecific binding thereof to specific hemozoin crystal faces and subsequent inhibition of crystal growth. The experimental evidence indicating adsorption of the quinoline type drugs to particular crystal faces is reviewed in terms of possible binding sites on those faces, the functional groups of the drug, change in crystal (β-hematin) morphology, and various methods, suggesting that the quinoline drugs were occluded within the growing crystals. The drugs may be described as hemozoin crystal capping agents (CMOLs) in view of their proposed ability to bind stereospecifically to particular faces of the crystal, despite being completely different in molecular structure from that of the β-hematin cyclic molecular dimer, and thus inhibit growth along two principal directions. In concert with pure drug, halofantrine or quinoline methanolFe3+-PPIX complexes would act as TMAs binding stereoselctively to the third principal face and inhibiting its growth. On the other hand, artemisinin drug action is described as a result of binding of artemisinin-β-hematin cyclic dimer adducts to all of the principal faces of hemozoin, resulting in effective crystal growth inhibition. Such proposed adducts may be considered as tailor-made crystal growth inhibitors since their molecular structures are the same as that of the β-hematin cyclic dimer but for an added (artemisinin) moiety bound to various sites on the periphery of the porphyrin rings and thus tailored for adsorption to specific faces. A detailed description of the various hemozoin surface binding sites with a focus on their spatial arrangement is given, to best design effective inhibitors of hemozoin nucleation and growth. Finally, nucleation of hemozoin crystals is reviewed in view of very recent findings that acyl-glycerols within lipid bodies promote the nucleation process. Future work would have to provide evidence whether there is any crystal alignment within the lipid bodies including the crystallographic nature thereof and develop methods to map the distribution of antimalarial drugs occluded within hemozoin crystals.

6. Acknowledgments We are deeply indebted to our former collaborators who have contributed to part of the studies reviewed here. Our work in this field was supported by the Kimmelmann Center at the Weizmann Institute of Science. We are thankful to Dr. Irena Efremenko in having performed the DFT computations on various molecular systems and to Dr. Yael DiskinPosner for conducting a search on the CSD.

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7. References (1) Uhlemann, A. C.; Krishna, S. In Drugs, Disease and Post-genomic Biology, Current Topics in Microbiology and Immunology; Sullivan, D. J., Krishna, S., Eds.; Springer-Verlag: Berlin, Heidelberg, 2005; Vol. 295. (2) Bouland, P. B. Drug Resistance in Malaria; World Health Organization: Switzerland, 2001. (3) Ziegler, J.; Linck, R.; Wright, D. W. Curr. Med. Chem. 2001, 8, 171. (4) Egan, T. J.; Mavuso, W. W.; Ncokazi, K. K. Biochemistry 2001, 40, 204. (5) Hempelmann, E.; Egan, T. J. Trends Parasitol. 2002, 18, 11. (6) Oaks, S. C., Jr., Mitchell, V. S., Pearson, G. W., Aarpenter, C. C. J., Eds. Malaria. Obstacles and Opportuinities; National Academy Press: Washington, DC, 1991. (7) Bray, P. G.; Ward, S. A.; O’Neill, P. M. In Drugs, Disease and Postgenomic Biology, Current Topics in Microbiology and Immunology; Sullivan, D. J., Krishna, S., Eds.; Springer-Verlag: Berlin, Heidelberg, 2005; Vol. 295. (8) Scholl, P. E.; Tripathi, A. K.; Sullivan, D. J. In Drugs, Disease and Post-genomic Biology, Current Topics in Microbiology and Immunology; Sullivan, D. J., Krishna, S., Eds.; Springer-Verlag: Berlin, Heidelberg, 2005; Vol. 295. (9) Goldberg, D. E. In Drugs, Disease and Post-genomic Biology, Current Topics in Microbiology and Immunology; Sullivan, D. J., Krishna, S., Eds.; Springer-Verlag: Berlin, Heidelberg, 2005; Vol. 295. (10) Wiesner, J.; Ortmann, R.; Jomaa, H.; Schlitzer, M. Angew. Chem., Int. Ed. 2003, 42, 5274. (11) Egan, T. J. Targets 2003, 2, 115. (12) Slater, A. F. G.; Swiggart, W. J.; Orton, B. R.; Flitter, W. D.; Goldberg, D. E.; Cerami, A.; Henderson, G. B. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 325. (13) Pagola, S.; Stephens, W. P.; Bohle, D. S.; Kosar, A. D.; Madsen, S. K. Nature 2000, 404, 307. (14) Oliveira, M. F.; Kycia, S. W.; Gomez, A.; Kosar, A. J.; Bohle, D. S.; Hempelmann, E.; Menezes, D.; Vannier-Santos, M. A.; Oliveira, P. L.; Ferreira, S. T. FEBS Lett. 2005, 579, 6010. (15) Sullivan, D. J.; Gluzman, I. Y.; Russell, D. G.; Goldberg, D. E. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11865. (16) Sullivan, D. J.; Matile, H.; Ridley, R. G.; Goldberg, D. E. J. Biol. Chem. 1998, 273, 31103. (17) Egan, T. J.; Ross, D. C.; Adams, P. A. FEBS Lett. 1994, 352, 54. (18) Egan, T. J.; Mavuso, W. W.; Ross, D. C.; Marques, H. M. J. Inorg. Biochem. 1997, 68, 137. (19) Hawley, S. R.; Bray, P. G.; Mungthin, M.; Atkinson, J. D.; O’Neill, P. M.; Ward, S. A. Antimicrob. Agents Chemother. 1998, 42, 682. (20) Dorn, A.; Vippagunta, S. R.; Matile, H.; Jaquet, C.; Vennerstrom, J. L.; Ridley, R. G. Biochem. Pharmacol. 1998, 55, 727. (21) Chen, M. M.; Shi, L.; Sullivan, D. J. Mol. Biochem. Parasitol. 2001, 113, 1. (22) Hartman, P.; Perdok, W. G. Acta Crystallogr. 1955, 8, 49. (23) Hartman, P.; Perdok, W. G. Acta Crystallogr. 1955, 8, 525. (24) Hartman, P. In North-Holland Series in Crystal Growth; Bardsley, W., Hurle, D. T. J., Mullin, J. B., Eds.; North-Holland Publishing Co.: Amsterdam, 1973; Vol. 1. (25) Hartman, P.; Bennema, P. J. Cryst. Growth 1980, 49, 145. (26) Buller, R.; Peterson, M. L.; Almarsson, O.; Leiserowitz, L. Cryst. Growth Des. 2002, 2, 553. (27) Noland, G. S.; Briones, N.; Sullivan, D. J. Mol. Biochem. Parasitol. 2003, 130, 91. (28) Bohle, D. S.; Kosar, A. D.; Stephens, W. P. Acta Crystallogr. 2002, D58, 1752. (29) Solomonov, I.; Osipova, M.; Feldman, Y.; Baehtz, C.; Kjaer, K.; Robinson, I. K.; Webster, G. T.; McNaughton, D.; Wood, B. R.; Weissbuch, I.; Leiserowitz, L. J. Am. Chem. Soc. 2007, 129, 2615. (30) Weissbuch, I.; Lahav, M.; Leiserowitz, L. Cryst. Growth Des. 2003, 3, 125. (31) DeVries, A. L. Philos. Trans. R. Soc. 1984, 304, 575. (32) Chakrabartty, A.; Yang, D. S. C.; Hew, C. L. J. Biol. Chem. 1984, 264, 11313. (33) Knight, C. A.; Chang, C. C.; DeVries, A. L. Biophys. J. 1991, 59, 409. (34) Weissbuch, I.; Addadi, L.; Lahav, M.; Leiserowitz, L. Science 1991, 253, 637. (35) Weissbuch, I.; Popovitz-Biro, R.; Lahav, M.; Leiserowitz, L. Acta Crystallogr. 1995, B51, 115. (36) Poser, C. M.; Bruyn, G. W. An Illustrated History of Malaria; The Parthenon Publishing Group: New York, London, 1999.

Weissbuch and Leiserowitz (37) Verdier, F.; Lebras, J.; Clavier, F.; Hatin, I.; Blayo, M. C. Antimicrob. Agents Chemother. 1985, 27, 561. (38) Fitch, C. D. Science 1970, 169, 289. (39) Moreau, S.; Perly, B.; Chachaty, C.; Deleuze, C. Biochimie 1982, 64, 1015. (40) O’Neill, P. M.; Willock, D. J.; Hawley, S. R.; Bray, P. G.; Storr, R. C.; Ward, S. A.; Park, B. K. J. Med. Chem. 1997, 40, 437. (41) Desiraju, G. R.; Steiner, T. The Weak Hydrogen Bond in Structural Chemistry and Biology; Oxford University Press: Oxford, NY, 1999. (42) de Villiers, K. A.; Marques, H. M.; Egan, T. J. J. Inorg. Biochem. 2008, 102, 1660. (43) Burgess, S. J.; Selzer, A.; Kelly, J. X.; Smilkstein, M. J.; Riscoe, M. K.; Peyton, D. H. J. Med. Chem. 2006, 49, 5623. (44) Wood, B. R.; Langford, S. J.; Cooke, B. M.; Lim, J.; Glenister, F. K.; Duriska, M.; Unthank, J. K.; McNaughton, D. J. Am. Chem. Soc. 2004, 126, 9233. (45) Kelly, J. X.; Winters, R.; Peyton, D. H.; Hinrichs, D. H.; Riscoe, M. Antimicrob. Agents Chemother. 2002, 46, 144. (46) Riscoe, M.; Kelly, J. X.; Winter, R. Curr. Med. Chem. 2005, 12, 2539. (47) Stead, A. M. W.; Bray, P. G.; Edwards, I. G.; Dekoning, H. P.; Elford, B. C.; Stocks, P. A.; Ward, S. A. Mol. Pharmacol. 2001, 59, 1298. (48) Meshnick, S. H. Int. J. Parasitol. 2002, 32, 1655. (49) Hong, Y. L.; Yang, Y. Z.; Meshnick, S. R. Mol. Biochem. Parasitol. 1994, 63, 121. (50) Pandey, A. V.; Tekwani, B. L.; Singh, R. L.; Chauhan, V. S. J. Biol. Chem. 1999, 274, 19383. (51) Robert, A.; Coppel, Y.; Meunier, B. Chem. Commun. 2002, 414. (52) Nader, F.; Leiserowitz, L. Acta Crystallogr. B 1977, 33, 3670. (53) Hawley, S. R.; Bray, P. G.; Park, B. K.; Ward, S. A. Mol. Biochem. Parasitol. 1996, 80, 15. (54) Hawley, S. R.; Bray, P. B.; O’Neill, P. M.; Park, P. K.; Ward, S. A. Biochem. Pharmacol. 1996, 52, 723. (55) Efremenko, I. Unpublished PBEO/sdd computations. (56) Berkovitch-Yellin, Z.; Leiserowitz, L. Acta Crystallogr. 1984, B40, 159. (57) Slouf, M.; Holy, A.; Petricek, V.; Cisarova, I. Acta Crystallogr. 2002, B58, 519. (58) Shanks, G. D.; Kain, K. C.; Keystone, J. C. Clin. Infect. Dis. 2001, 33, 381. (59) Jacquemain, D.; Wolf, S. G.; Leveiller, F.; Frolow, F.; Eisenstein, M.; Lahav, M.; Leiserowitz, L. J. Am. Chem. Soc. 1992, 114, 9983. (60) Berkovitch-Yellin, Z.; Leiserowitz, L. Acta Crystallogr. B 1977, 33, 3670. (61) Berkovitch-Yellin, Z.; Leiserowitz, L. J. Am. Chem. Soc. 1977, 99, 6106. (62) Ridley, R. G.; Holheinz, W.; Matile, H.; Aquet, C.; Dom, A.; Masciardi, R.; Jolidon, S.; Richter, W. F.; Guenzi, A.; Girometta, M.A.; Urwyler, H.; Huber, W.; Haithong, S. T.; Peters, W. Antimicrob. Agents Chemother. 1996, 40, 1846. (63) IC50 (inhibitory concentration 50) is the concentration of a compound needed to reduce the population growth of organisms. (64) deVries, P. J.; Birch, N. N.; Thien, H. V.; Hung, L. N.; Anh, T. K.; Kager, P. A.; Heisterkamp, S. H. Antimicrob. Agents Chemother. 2000, 44, 1302. (65) Gupta, S.; Thapar, M. M.; Mariga, S. T.; Wernsdorfer, W. H.; Bjorkman, A. Exp. Parasitol. 2002, 100, 28. (66) Olliaro, P. L.; Taylor, W. R. J. Postgrad. Med. 2004, 50, 40. (67) Goldberg, D. E.; Slater, A. F. G.; Cerami, A.; Henderson, G. B. Proc. Natl Acad. Sci. U.S.A. 1990, 87, 2931. (68) Sullivan, D. J.; Gluzman, I. Y.; Goldberg, D. E. Science 1996, 271, 219. (69) Egan, T. E. J. Inorg. Biochem. 2002, 91, 19. (70) Bendrat, K.; Berger, B. J.; Cerami, A. Nature 1995, 378, 138. (71) Jackson, K. E.; Klonis, N.; Ferguson, D. J. P.; Adisa, A.; Dogovski, C.; Tilley, L. Mol. Microbiol. 2004, 54, 109. (72) Palacpac, N. M. Q.; Hiramine, Y.; Mi-ichi, F.; Torii, M.; Kita, K.; Hiramatsu, R.; Horii, T.; Mitamura, T. J. Cell Sci. 2004, 117, 1469. (73) Egan, T. J. J. Inorg. Biochem. 2008, 102, 1288. (74) Pisciotta, J. M.; Coppens, I.; Tripathi, A. K.; Scholl, P. F.; Schuman, J.; Bajad, S.; Shulaev, V.; Sullivan, D. J. Biochem. J. 2007, 402, 197. (75) Trager, W. Trends Parasitol. 2003, 19, 388. (76) Fitch, C. D.; Cai, G. Z.; Shen, Y. F.; Shoemaker, J. D. Biochim. Biophys. Acta, Mol. Basis Dis. 1999, 1454, 31. (77) Egan, T. J.; Chen, J. Y.-J.; de Villiers, K. A.; Mabotha, T. E.; Naidoo, K. J.; Ncokazia, K. K.; Langford, S. J.; McNaughton, D.; Pandiancherri, S.; Wood, B. R. FEBS Lett. 2006, 580, 5105.

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