Artemisinin, an Endoperoxide Antimalarial, Disrupts the Hemoglobin Catabolism and Heme Detoxification Systems in Malarial Parasite*

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 27, Issue of July 2, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Artemisinin, an Endoperoxide Antimalarial, Disrupts the Hemoglobin Catabolism and Heme Detoxification Systems in Malarial Parasite* (Received for publication, September 16, 1998, and in revised form, April 13, 1999) Amit V. Pandey, Babu L. Tekwani, Ram L. Singh, and Virander S. Chauhan From the Malaria Research Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, P. O. Box 10504, New Delhi , the Division of Biochemistry, Central Drug Research Institute, Lucknow , and the Department of Biochemistry, R.M.L. Avadh University, Faizabad , India Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well as Plasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains. The fast spreading resistance to commonly used quinoline antimalarials has made malaria a major global disease (1). Among the few alternative drugs available, artemisinin, an endoperoxide antimalarial drug, derived from an ancient Chinese herbal remedy, Qinghaosu, is the most promising (2, 3). A preparation containing racemic mixture of - and -arteether (an analogue of artemisinin) has successfully completed clinical trials in India (4). However, very little is known about the molecular mechanism of action of these drugs. A two-step mechanism proposed recently by Meshnick et al. (5) suggests the heme-catalyzed cleavage of the endoperoxide bridge forms a free radical followed by specific and selective alkylation of some malarial proteins (6). However, this does not explain the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, P. O. Box 10504, New Delhi , India. Tel.: ; Fax: ; This paper is available on line at selective cytotoxic action of artemisinin on mature parasite stages (late stage trophozoites and schizonts) (7, 8). The chloroquine-resistant strains of Plasmodium berghei that lack hemozoin are also resistant to artemisinin, indicating that the presence of preformed hemozoin may be necessary for antimalarial action of these drugs (9). During intraerythrocytic development and proliferation, hemoglobin is utilized as a major source of amino acids by the malarial parasite (10 12). Constant degradation of hemoglobin inside the parasite food vacuole occurs through a sequentially ordered process that involves cysteine as well as aspartic acid proteases (13 16). The toxic free heme, which is generated due to digestion of hemoglobin, is simultaneously detoxified by the malarial parasite through a specific mechanism of heme polymerization (17 20). The polymerized heme commonly referred to as hemozoin or malaria pigment accumulates in the form of a crystalline, insoluble, black-brown pigment (21 23). Once the parasite life cycle is complete, this pigment is sequestered to various tissues of the host (24). The heme polymerization pathway is specific to the malarial parasite and offers a potential biochemical target for the design of antimalarials (25). An enzyme heme polymerase was initially described, which could promote hemozoin formation (26 28), but the molecular mechanisms of this process are still under debate (29 37). Recent studies have shown that artemisinin taken up by the malarial parasite growing in vitro was selectively concentrated in the parasite food vacuole and was associated with hemozoin (38). Artemisinin also interacts with heme, forming covalent adducts (38, 39). In this report, we describe the inhibition of hemoglobin breakdown and heme polymerization by artemisinin and one of its analogues -/ -arteether. Our studies provide evidence that the antimalarial effect of artemisinin may at least partly be due to inhibition of malarial hemoglobin degradation pathway and heme detoxification system. EXPERIMENTAL PROCEDURES Reagents [ 14 C]Leucine was a kind gift from Prof. O. P. Shukla (Central Drug Research Institute, Lucknow, India). Peptide substrate Z 1 -FR-AMC was purchased from Bachem. Artemisinin and -/ -arteether were kindly provided by Dr. S. K. Puri. Bovine serum albumin standard solution was from Pierce, and Ni 2 -NTA-Sepharose was from Amersham Pharmacia Biotech (Sweden). Hemin, SDS, dithiothreitol, isopropyl-1-thio- -D-galactopyranoside, E-64, pepstatin A, LB media, and all other chemicals were from Sigma. Parasite and Experimental Host Male Swiss albino mice (obtained from the Division of Laboratory Animals, Central Drug Research Institute, Lucknow, India) weighing g were infected with Plasmodium 1 The abbreviations used are: Z, benzyloxycarbonyl; AMC, aminomethylcoumarin; PBS, phosphate-buffered saline; PfHRP II, Plasmodium falciparum histidine-rich protein II; TCTP, translationally controlled tumor protein.

2 19384 Artemisinin and Heme Detoxification by Plasmodium yoelii nigeriensis by intraperitoneal passage of infected erythrocytes. Parasitemia was monitored by microscopic examination of Giemsa-stained thin blood smears. Blood was collected at high levels of parasitemia ( 60%) in sterile acid/citrate/dextrose. Animals were housed in the animal house at the Institute, and care was provided as per the guidelines laid down by ethics committee. Preparation of 14 C-Labeled Hemoglobin Swiss albino mice were injected with phenylhydrazine (40 mg/kg body weight) to induce reticulocytes. Blood was collected 3 days post-phenylhydrazine injection. 50 Ci of [ 14 C]leucine was added to this and incubated at 37 C for 10 h in a conical flask rotating at 60 rpm. Erythrocytes were pelleted by centrifugation; leukocytes were removed, and hemoglobin was purified as described previously (12). Assay of Food Vacuole Proteases Blood from 30 mice infected with P. yoelii was pooled and centrifuged at 500 g for 10 min at 4 C. The plasma and buffy coat were removed, and the erythrocyte pellet was suspended in phosphate-buffered saline (PBS) and passed through a CF-11 column to remove leukocytes. Food vacuole preparation was done as described previously (12). Purified vacuoles were resuspended in acetate buffer (ph 5.0, 100 mm) and homogenized by a Polytron homogenizer at 6000 rpm. This vacuole extract was used for the protease assay. Proteolytic activity was measured using 14 C-labeled bovine hemoglobin as a substrate. Reactions were performed in a total volume of 1.0 ml containing 50 l of vacuolar lysate, 100 g of hemoglobin, 1 mm CaCl 2, 1 mm dithiothreitol in 100 mm acetate buffer (ph 5.0). The reaction was stopped by adding 500 l of chilled 15% trichloroacetic acid, and the radioactivity in trichloroacetic acid-soluble fractions was monitored as a measure of proteolytic activity. Fluorimetric assay of cysteine protease was performed on an LS 50B spectrofluorimeter from Perkin-Elmer using Z-Phe-Arg-AMC as substrate as described previously (14). Fluorescence of aminomethylcoumarin (AMC) released by proteolysis was used to monitor the activity. All assays using peptide substrates were performed at 25 C in a continuous manner for 3 min total time. Interaction of Artemisinin with Substrate Artemisinin was incubated with hemoglobin to study the drug substrate interaction. Hemoglobin was extensively dialyzed after artemisinin exposure and checked by electrophoresis. Effect of Artemisinin on Parasite Hemoglobin Catabolism The drug was incubated with infected erythrocytes for 0 6 h at 37 C. After the incubation erythrocytes were washed with PBS, and parasites were collected by saponin lysis. Parasites were lysed by the addition of SDS-polyacrylamide gel electrophoresis sample buffer and boiling for 3 min and kept at 20 C until further use. SDS-Polyacrylamide Gel Electrophoresis Analysis Electrophoresis was carried out according to standard protocols. Gels contained 15% acrylamide and were stained with Coomassie Blue for protein visualization. Bovine hemoglobin was used as a control. Purification and Characterization of Hemozoin Hemozoin was purified from the erythrocytes of the mice infected with P. yoelii according to the method described earlier (24). Preparation of Recombinant Histidine-rich Protein II Plasmid containing the gene encoding P. falciparum HRP II (PfHRP II) in a pet 3d vector (Novagen) was a kind gift from Dr. D. E. Goldberg (Washington University, St. Louis, MO). Plasmids were transformed into Escherichia coli strain BL21(DE3). E. coli cells containing the plasmid were grown at 37 C until A 600 reached between 0.4 and 0.6 and were cooled to 30 C, and PfHRP II expression was induced by the addition of isopropyl-1-thio- -D-galactopyranoside to a final concentration of 0.4 mm. Cells were allowed to grow for 8 h with shaking at 30 C. Purification of PfHRP II was performed by metal chelate chromatography on Ni 2 -NTA-Sepharose followed by HPLC (Waters) on a reverse phase C 18 Bondapak column. In Vitro Heme Polymerization Assay Heme polymerization activity was assayed using P. yoelii lysates or PfHRP II (40, 41). For preparing parasite lysate, plasma and buffy coat were removed from the infected blood, and the erythrocyte pellet was washed once with PBS and suspended in 4 volumes of PBS containing glucose (0.9% w/v). The lysate was prepared by freezing the suspension in liquid nitrogen. Frozen droplets of the lysate were stored in aliquots at 70 C until further use. When required, an aliquot of the lysate was thawed and centrifuged at 16,000 g for 20 min at 4 C. The pellet was resuspended in acetate buffer (100 mm, ph 5.0) by brief sonication and used for heme polymerization assay. The assay mixture contained 50 l of the parasite extract, 100 M hemin as the substrate and acetate buffer (100 mm, ph 5.0) in a total volume of 1.0 ml. Two controls, one lacking the substrate and the other lacking the parasite extract, but containing only 100 M hemin in the acetate buffer, were run simultaneously. Each assay was TABLE I Inhibition of P. yoelii vacuolar proteolytic activity by artemisinin using denatured hemoglobin as substrate Concentration of inhibitors was as follows: artemisinin 200 M, E M, pepstatin A 100 M, heme 20 M. Assay was performed as described under Experimental Procedures. Inhibitors were incubated with enzyme for 1 h before addition of substrate. Values are mean S.D. of triplicate observations. Substrate no. Sample dpm Inhibition % 1 Control Artemisinin Arteether E Heme E-64 artemisinin Pepstatin A Pepstatin A artemisinin Pepstatin A E set up in triplicate and incubated at 37 C for 4 h (16 h for assays using PfHRP II) in a constantly shaking water bath. The reaction was stopped by centrifugation at 16,000 g for 5 min, and pellets were resuspended in Tris-HCl (100 mm, ph 7.4) containing 2.5% SDS. The pellets were washed twice with same buffer and once with sodium bicarbonate buffer (100 mm, ph 9.0). The final pellet thus obtained was of polymerized heme (hemozoin). For quantitation of the hemozoin, the pellets were solubilized in 50 l of2n NaOH, and spectra were recorded, using 2.5% SDS as solvent and blank, in the range of nm on a Hitachi-557 double beam spectrophotometer. An extinction coefficient of 91,000 M 1 cm 1 at 400 nm was used to quantitate the hemozoin in the form of heme as described previously (42). In some experiments instead of parasite lysate, recombinant PfHRP II was used as a template for hemozoin formation. Interaction of Artemisinin and -/ -Arteether with Hemozoin Interaction of drugs with malarial hemozoin was studied as described previously (43). A suspension of fine crystals of purified hemozoin was prepared by sonication. Hemozoin was incubated in acetate buffer (ph 5.0, 100 mm, final volume 1.0 ml) for 4hat37 Cwith the specified concentration of artemisinin or -/ -arteether. Controls without the drug were also run simultaneously, and each assay was run in triplicate. At the end of the incubation period, the suspensions were centrifuged at 10,000 g for 5 min. The hemozoin pellets were washed once with ethanol to remove the drugs and once with alkaline bicarbonate buffer (ph 9.0, 100 mm). The amount of hemozoin remaining was quantified as described previously (27). The difference between amount of hemozoin in the incubation mixtures without and with the drug gave the amount of hemozoin breakdown due to the endoperoxide antimalarials. Protein Estimation Protein was estimated by the method of Lowry et al. (44) using bovine serum albumin as standard. RESULTS Inhibition of Hemoglobin Degradation To study the effect of artemisinin and its analogue arteether on the hemoglobin catabolic process of the malarial parasite, we used purified digestive vacuoles from P. yoelii as the source of proteolytic activity. We found that artemisinin inhibited the proteolytic activity of the digestive vacuole lysate in an in vitro assay, involving degradation of 14 C-labeled hemoglobin (Table I). Around 70% of the proteolytic activity in malaria food vacuole is due to aspartic acid proteases (13). After treatment of the vacuolar lysate with pepstatin A (a specific inhibitor of aspartic acid proteases), 71% inhibition of the proteolytic activity was observed. The remaining proteolytic activity ( 29%) in the reaction mixture could be inhibited by E-64 (a highly specific cysteine protease inhibitor). Artemisinin treatment also resulted in similar inhibition. Maximum inhibition caused by artemisinin was comparable to that achieved by E-64, suggesting the specific inhibition of cysteine protease by this drug. Addition of artemisinin in reaction mixture after E-64 treatment did not result in any significant addition into the inhibition of proteolysis as compared with E-64 alone. This indicated

3 TABLE II Inhibition of P. yoelii cysteine protease activity by artemisinin Activity was measured by degradation of specific peptide for plasmodial cysteine protease. Concentration of inhibitors was as follows: artemisinin 50 M, E M, pepstatin A 100 M, heme 1 M. Assay was performed as described under Experimental Procedures. Inhibitors were incubated with enzyme for 1 h before addition of substrate. Artemisinin and Heme Detoxification by Plasmodium Substrate no. Sample Fluorescence intensity (arbitrary units)/ min 1 Control Artemisinin (preincubated with 19 3 enzyme) 3 Artemisinin (added before start) Artemisinin heme 11 5 (preincubation with enzyme) (heme mm) 5 Heme (preincubated with enzyme) 32 6 (heme mm) 6 E Pepstatin A 34 5 that the specific protease targets of both E-64 and artemisinin might be same. On the other hand, combination of either pepstatin A and E-64 or pepstatin A and artemisinin resulted in almost complete inhibition of the vacuolar proteolytic activity. This was expected as both aspartic and cysteine proteases would be blocked by these inhibitors. These results suggested that artemisinin may be involved in specific inhibition of malarial cysteine protease activity. We next investigated whether the observed inhibition could be due to the specific interaction of artemisinin with the parasite cysteine protease. For this a fluorogenic peptide substrate of malarial cysteine protease, Z-Phe-Arg-AMC, was used to study the mechanism of protease inhibition by artemisinin. In a continuous fluorometric assay when artemisinin was incubated with the parasite lysate for 1 h prior to the assay, significant decrease in the proteolytic cleavage of peptide substrate was observed indicating that the drug could be making some modifications in the enzyme (Table II). The inhibition was significantly higher when heme was included in the reaction mixture along with artemisinin. No change in the protease activity was observed when artemisinin was added just before the start. To confirm whether artemisinin could inhibit the hemoglobin degradation inside the malarial parasite, artemisinin was incubated in vitro with P. yoelii-infected erythrocytes for 0 6 h, and the hemoglobin levels in the cell-free parasite preparations were monitored by SDS-polyacrylamide gel electrophoresis. We observed that artemisinin-treated parasites showed a higher level of hemoglobin compared with the untreated parasites (Fig. 1). These results suggest that degradation of hemoglobin in the digestive vacuole of the malarial parasite may have been inhibited by artemisinin treatment. Effect of Endoperoxides on Heme Polymerization in Vitro When free heme is incubated with the particulate fraction of the cell-free P. yoelii under conditions similar to that of the parasite food vacuole, a fraction of heme is polymerized, which can be identified as a product insoluble in SDS (2.5% w/v) and alkaline bicarbonate solution (41). However, no such product was formed when heme alone (without any parasite extract) was processed in a similar manner (29). We found that the formation of hemozoin by P. yoelii extract is dependent on incubation time, amount of protein, and concentration of free heme (41). A similar pattern has been observed by earlier workers (26, 27) for heme polymerization by P. falciparum and P. berghei. Recently, a histidine-rich protein (PfHRP II) from P. falciparum has been shown to catalyze polymerization of heme FIG. 1. SDS-polyacrylamide gel electrophoresis analysis of proteins in P. yoelii parasites after artemisinin exposure. A, molecular weight markers (lane 1), control parasites (lane 2), parasites exposed to artemisinin (lane 3), and hemoglobin (lane 4). B, parasites after 0 h exposure (lane 1),1(lane 2),2(lane 3),4(lane 4),and6hof exposure (lane 5). Numbers on the left indicate positions of the molecular weight markers (M r 10 3 ). Other conditions of the experiments were as described under Experimental Procedures. (40). We also used recombinant PfHRP II as a source of heme polymerization activity in one of the experiments to study the effect of artemisinin on hemozoin formation in vitro. Artemisinin as well as -/ -arteether caused marked inhibition of heme polymerization mediated by the parasite lysate (Fig. 2A) as well as PfHRP II (Fig. 2B). Inhibition of hemozoin formation by -/ -arteether (IC 50 value 7.3 M) was slightly higher than that by artemisinin (IC 50 value 11.5 M) (Fig. 2A). The effect of increasing substrate (Fig. 3A) and inhibitor (Fig. 3B) concentration on inhibition of heme polymerization was also studied. Normally polymerization of heme in the presence of parasite extract or PfHRP II is a saturable reaction following a hyperbolic pattern (41). However, the inhibition of heme polymerization by artemisinin was not affected by the presence of high concentrations of heme to any significant extent; following an initial fall in the percent inhibition between 5 and 10 M, the inhibition potency of drug remained almost constant up to 200 M heme (Fig. 3A). The pattern of inhibition was qualitatively similar at 1 and 10 M concentrations of artemisinin with the expected quantitative increase for higher drug concentration. A K i value of 12.0 M was obtained for inhibition of P. yoelii-mediated heme polymerization by artemisinin, from the Dixon plot (Fig. 3B). Interaction of Endoperoxides with Malarial Hemozoin in Vitro Studies on the interaction of purified hemozoin with artemisinin and -/ -arteether revealed a novel reaction. The incubation of purified hemozoin with artemisinin or -/ -arteether under acidic conditions (acetate buffer, 100 mm, ph 5.0) resulted in the loss of hemozoin contents as compared with control, indicating that hemozoin may be disrupted as a result of its interaction with the endoperoxide. This hemozoin disruption increased with the increasing concentration of the drug (Fig. 4). Interaction of hemozoin with the drug may result in breakdown of the hemozoin pigment which could then form a complex with the heme units (38). DISCUSSION Earlier Meshnick et al. (5) proposed a two-step mechanism for the antimalarial action of artemisinin and other related endoperoxides. In the first step, the endoperoxide bridge in artemisinin is cleaved by free heme or iron, leading to the generation of an unstable radical of the drug. This subse-

4 19386 Artemisinin and Heme Detoxification by Plasmodium FIG. 2.Dose-dependent inhibition of heme polymerization by endoperoxide antimalarial drugs. Effect of artemisinin (E) and -/ -arteether ( ) on polymerization of heme in vitro by P. yoelii extracts (A) and inhibition by artemisinin of PfHRP II-mediated heme polymerization (B). Each point represents mean S.D. of triplicate observations. FIG. 3.Inhibition of heme polymerase activity of P. yoelii extracts by artemisinin at varying substrate concentrations (A) and varying concentration of the drug (B). A, Lineweaver-Burk plot of the reaction containing 0 (E), 1.0 M ( ), and 10.0 M ( ) inhibitor (artemisinin) and variable concentrations of the substrate (heme). B, Dixon plot of inhibition containing 50 M ( ) or100 M (E) of substrate (heme) and variable concentrations of inhibitor (artemisinin). The values are mean S.D. of triplicate observations. quently causes selective alkylation of malarial proteins, leading to death of the parasite. However, the transient source of free heme or iron proposed in this mechanism has not been clearly described. The heme released as a result of hemoglobin degradation is simultaneously polymerized to form hemozoin, and the malarial parasite still depends on the extracellular source of iron to fulfill its nutritional requirements. The results presented in this communication clearly demonstrate that the endoperoxide antimalarials interrupt the hemoglobin catabolism system of the malarial parasite by causing inhibition of hemoglobin degradation as well as polymerization of heme to hemozoin. Our results also show that inhibition of heme polymerization by endoperoxide antimalarials is more potent than the quinoline antimalarials as reported earlier (45). Also, unlike artemisinin, quinoline antimalarials are poor inhibitors of malarial digestive vacuole proteases responsible for hemoglobin degradation. Like quinoline antimalarials, [ 14 C]artemisinin and [ 3 H]dihydroartemisinin have been shown to be selectively concentrated by the Plasmodium-infected erythrocytes, and the drugs are predominantly associated with the malaria pigment (38). Endoperoxide antimalarials are fast acting drugs, which exert their antimalarial effect within an hour of administration (7). These drugs have a highly selective effect on different stages of the malarial parasite and mainly affect the mature stages of the parasite (late trophozoites), which are heavily laden with hemozoin pigment. Artemisinin is ineffective against those parasite strains that do not produce the malaria pigment (9). Disruption of heme polymerization or inhibition of vacuolar proteases, although important targets for the anti-parasitic activity of the drug, may not be sufficient to explain the selective and fast antimalarial action of artemisinin. The endoperoxides additionally initiate the breakdown of the malaria pigment already present in the parasite food vacuole. This reaction could easily lead to a rapid increase in the indigenous heme level, which may not be detoxified by the usual heme polymerization pathway since this activity is already blocked by artemisinin. Our results point toward a threestep effect of endoperoxide drugs on malarial parasite. Inhibition of hemoglobin degradation as well as heme polymerization would start accumulation of heme, which could be further assisted by direct interaction of the drug with malaria pigment already present inside the digestive vacuole. This may be a major reason for the fast action of these drugs compared with quinoline antimalarials. Since artemisinin has been shown to form complexes with heme (38), it is conceivable that its interaction with hemozoin may initiate a process of molecular rearrangement leading to the breakdown of iron carboxylate bond of hemozoin, which links different heme units, thus releasing heme from the hemozoin. Specific interaction of endoperoxide drugs on three distinct steps in hemoglobin digestion and related heme detoxification pathways of malarial parasite would suggestively lead to a fast build up of a pool of free heme and thus would explain the generation of a transient source of heme which is necessary for the antimalarial action of artemisinin and related endoperoxide antimalarials.

5 Artemisinin and Heme Detoxification by Plasmodium FIG. 4.Effect of artemisinin and -/ -arteether in vitro on purified hemozoin. Each bar shows values (mean S.D.) of triplicate observations. FIG. 5. A schematic diagram describing the proposed blood schizontocidal mechanism of action of artemisinin and related endoperoxide antimalarials. The precise mechanism of hemozoin formation by the malarial parasite is still unclear (25). However, recently published results by us and other groups (34 36) show that under physiological conditions heme polymerization could not occur as a spontaneous chemical process. Our studies on heme polymerization under different experimental conditions demonstrate that no hemozoin could be formed in the absence of malarial parasite extract (34, 41). A product formed spontaneously in the absence of parasite material has been characterized by us and other workers (22, 31, 34) as a heme-acetate adduct that may be formed by the linking of a central ferric iron of heme and a carboxylate group of acids in the buffer used for the assay of heme polymerization. However, these products could still be differentiated by their solubility characteristics in SDS/bicarbonate buffer (21, 22, 24, 34). Fitch and Chou (36) have recently shown that no spontaneous heme polymerization occurs under any concentration of heme or acetate. In their report, a heatlabile as well as a heat-stimulated heme polymerase activity were characterized in the extracts of a rodent malarial parasite P. berghei. The histidine-rich protein localized in the digestive vacuoles of P. falciparum could provide the nucleus for the polymerization of heme (40). Recently we have shown that synthetic peptides based on a repetitive hexapeptide sequence (Ala-His-His-Ala-Ala-Asp) present in histidine-rich protein II from P. falciparum bind heme and inhibit hemozoin formation in vitro (41). These repeat sequences may provide a nucleation site for initiation of hemozoin formation. The mechanism of artemisinin accumulation inside the malarial digestive vacuole is not known. All the blood schizontocidal antimalarials bind to heme (45, 46). It has been proposed that binding with heme will lead to the accumulation of drugs in the parasite vacuole as the heme-bound form of the drug may not be able to cross the vacuolar membranes (39, 47). Artemisinin also binds to heme, forming covalent complexes (38), which may be the mechanism of its accumulation and toxicity in the parasite food vacuole. However, it has also been reported that the artemisininheme complex does not possess any antimalarial activity (39). This may be because formation of the complex in situ is accompanied by other reactions like free radical generation. Several changes occur during heme-artemisinin complex formation, and a fraction belonging to either of the components may be lost during the formation of artemisinin-heme complex in vitro; mass spectrum analysis of the isolated artemisinin-heme complex showed a value of mass less than the sum of artemisinin and heme (39). Also, it is quite likely that the preformed hemeartemisinin complex may not be able to reach the site of action (i.e. parasite digestive vacuole). Uptake of the preformed complex by the parasite has not yet been studied. Based on the information available so far, we have proposed a tentative scheme for the mode of action of artemisinin and related endoperoxide antimalarials (Fig. 5). Reactions like free radical generation and protein alkylation not shown by quinoline antimalarials might explain the better efficacy of artemisinin. An efficient heme-binding endoperoxide may also have better antimalarial properties by accumulating in larger concentrations at the site of drug action. Several proteins have been identified in malarial parasite that could bind artemisinin. One of these proteins is the P. falciparum translationally controlled tumor protein (TCTP) homolog (48). The TCTP was reported to react with artemisinin in situ and in vitro in the presence of hemin and as well as binding to heme itself. The function of the malarial TCTP and the role of this reaction in the mechanism of action of artemisinin await elucidation. On the other hand dihydroartemisinin, an analogue of artemisinin, has been reported to bind hemoglobin-h resulting in the ineffectiveness of the drug in malarial parasites residing in -thalassemic erythrocytes (49, 50). Molecular modeling studies of heme and artemisinin suggest that in the most stable docked configuration, the endoperoxide bridge of artemisinin is in close proximity to the central iron of heme. In contrast an inactive analogue of artemisinin, deoxyartemisinin, docks in a different manner (51, 52). Similar studies with synthetic endoperoxides suggest that docking between an active trioxane and the receptor, heme, is the crucial step for the drug action (53, 54). In these studies, peroxide bond of the trioxane is found to lie close to the central iron atom of heme, suggesting it to be an important criterion for parasiticidal action. Recently degradation of heme in malarial parasite has also been reported to account for part of the heme detoxification efforts by Plasmodia (55 57). It has been suggested that a major part of the free heme may be detoxified through the degradation mechanism. Although the degradation of heme in the digestive vacuole may be possible, it is quite unlikely that the glutathione, present in the parasite cytoplasm, may have a

6 19388 Artemisinin and Heme Detoxification by Plasmodium role under normal circumstances in heme detoxification. Damage to parasite membranes during the heme interaction with vacuolar membranes itself may be sufficient to kill the parasite. However, excess glutathione in the resistant strains of the parasite may be responsible for protecting it from the toxicity of heme-drug complexes or heme alone which may leach out due to the rupture of vacuolar membrane. More studies in this direction are needed before a role of glutathione in heme detoxification could be established. It would be interesting to study the role of artemisinin in heme detoxification through peroxidative or glutathione-mediated degradation. Since chloroquine resistance is related to decreased accumulation of the drug, artemisinin, which appears to have the same target but a different structure, may defy the factors responsible for the chloroquine resistance. Moreover, due to additional benefits of free radical generation by interaction with heme inside the food vacuole and inhibition of hemoglobin-degrading proteases, endoperoxide compounds are better prospects for antimalarial drug design (58 60). Further studies related to structural and mechanistic aspects of the interaction of endoperoxides with hemoglobin catabolic pathway of the malaria parasite may yield important information for the design of better antimalarials. Acknowledgments The help of Drs. S. K. Puri and Naresh Singh (Central Drug Research Institute, Lucknow, India) for providing P. yoelii; Kailash Pandey (International Centre for Genetic Engineering and Biotechnology, New Delhi, India) for hemoglobin gel assays; Dr. Amos Gaikwad for the digital photographs; and Dr. David Sullivan (The John Hopkins University) for helpful suggestions in expression and purification of recombinant PfHRP II is gratefully acknowledged. We thank Dr. D. E. Goldberg (Washington University) for providing plasmids containing HRP II gene. We express our appreciation to Drs. Chetan Chitnis, Shahid Jameel, Raj Bhatnagar, and Sanjay Singh (International Centre for Genetic Engineering and Biotechnology) and V. C. Pandey (Central Drug Research Institute) for their critical evaluation of this manuscript and helpful discussion and suggestions. REFERENCES 1. Su, X., Kirkman, L. A., Fujioka, H., and Wellems, T. E. (1997) Cell 91, Klayman, D. L. (1985) Science 228, Hien, T. T., and White, N. J. (1993) Lancet 341, Mohapatra, P. K., Khan, A. M., Prakash, A., Mahanta, J., and Srivastava, V. K. (1996) Ind. J. Med. Res. 104, Meshnick, S. R., Taylor, T. E., and Kamchonwongpaisan, P. (1996) Microbiol. Rev. 60, Yang, Y. Z., Asawamahasakda, W., and Meshnick, S. R. (1993) Biochem. Pharmacol. 46, Geary, T. G., Divo, A. A., and Jensen, J. B. (1989) Am. J. Trop. Med. Hyg. 40, Caillard, V., Beaute-Lafitte, A., Chabaud, A. G., and Landau, I. (1992) Exp. 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