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Antimicrob Agents Chemother. Oct 2009; 53(10): 4080–4085.
Published online Aug 3, 2009. doi:  10.1128/AAC.00088-09
PMCID: PMC2764187

The Proteasome Inhibitor Epoxomicin Has Potent Plasmodium falciparum Gametocytocidal Activity[down-pointing small open triangle]

Abstract

Malaria continues to be a major global health problem, but only a limited arsenal of effective drugs is available. None of the antimalarial compounds commonly used clinically kill mature gametocytes, which is the form of the parasite that is responsible for malaria transmission. The parasite that causes the most virulent human malaria, Plasmodium falciparum, has a 48-h asexual cycle, while complete sexual differentiation takes 10 to 12 days. Once mature, stage V gametocytes circulate in the peripheral blood and can be transmitted for more than a week. Consequently, if chemotherapy does not eliminate gametocytes, an individual continues to be infectious for several weeks after the clearance of asexual parasites. The work reported here demonstrates that nanomolar concentrations of the proteasome inhibitor epoxomicin effectively kill all stages of intraerythrocytic parasites but do not affect the viability of human or mouse cell lines. Twenty-four hours after treatment with 100 nM epoxomicin, the total parasitemia decreased by 78%, asexual parasites decreased by 86%, and gametocytes decreased by 77%. Seventy-two hours after treatment, no viable parasites remained in the 100 or 10 nM treatment group. Epoxomicin also blocked oocyst production in the mosquito midgut. In contrast, the cysteine protease inhibitors epoxysuccinyl-l-leucylamido-3-methyl-butane ethyl ester and N-acetyl-l-leucyl-l-leucyl-l-methioninal blocked hemoglobin digestion in early gametocytes but had no effect on later stages. Moreover, once the cysteine protease inhibitor was removed, sexual differentiation resumed. These findings provide strong support for the further development of proteasome inhibitors as antimalaria agents that are effective against asexual, sexual, and mosquito midgut stages of P. falciparum.

The current recommended treatments for malaria caused by Plasmodium falciparum, including artemisinin combination therapy, eliminate intraerythrocytic asexual parasites that are responsible for the clinical symptoms. However, these treatments do not kill mature intraerythrocytic gametocytes that are required for the transmission of the parasite (24). In contrast to the 2-day asexual cycle of P. falciparum, the production of a mature stage V gametocyte takes 10 to 12 days. Once mature gametocytes are taken up by a mosquito during a blood meal, fertilization is stimulated. The resulting zygotes develop into oocysts where thousands of sporozoites are produced that can be transmitted to humans during a subsequent blood meal. The prolonged period required for P. falciparum gametocyte maturation in the human host suggests that malaria can be transmitted for several weeks after asexual parasites are eliminated (23). Thus, the development of drugs that are effective against both asexual-stage parasites and gametocytes may directly decrease malaria morbidity and mortality and reduce the spread of the disease.

Cysteine protease and proteasome inhibitors have been found to affect asexual intraerythrocytic parasites and are being evaluated as possible antimalarial agents (4, 7, 10, 15, 19-21, 25). However, their effect on the 10- to 12-day course of intraerythrocytic gametocyte development has not been reported. Proteasome inhibitors also have not been tested on parasites taken up by a mosquito, while cysteine protease inhibitors have been shown to significantly decrease P. falciparum gamete surface antigen processing, oocyst production, and sporozoite maturation (7, 10). The dual cysteine and serine protease inhibitors l-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) also have been reported to reduce P. falciparum exflagellation and the transmission of Plasmodium berghei to mosquitoes (22, 26).

Genes predicted to code for cysteine proteases and the proteasome are expressed throughout gametocytogenesis, providing targets for both classes of compounds (12, 28). Falcipain 1 and the P. berghei orthologs of PfSERA8 (PbECP1) and metacaspase 1 (PbMC1) are the only proteases whose function has been studied directly during gametocytogenesis by targeted gene disruption (3, 9, 14). The disruption of falcipain 1 and PfECP1 affected oocyst production in the mosquito midgut but not the asexual or sexual intraerythrocytic stage, while no stage of the life cycle was affected by the PbMC1 knockout. The work described here evaluates the effect of cysteine and proteasome inhibitors during P. falciparum sexual differentiation and development in the mosquito midgut.

MATERIALS AND METHODS

Reagents.

Reagents were obtained from Sigma (St. Louis, MO) unless otherwise indicated and include N-acetyl-l-leucyl-l-leucyl-l-methioninal (ALLM), N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN), dimethyl sulfoxide (DMSO), epoxysuccinyl-l-leucylamido-3-methyl-butane ethyl ester (E64d), and TPCK. Morpholine urea-leucine-homophenyl-aldehyde (MLHF) was obtained from MP Biomedicals (Solon, OH), and epoxomicin was from Sigma or Biomol (Enzo Biochem, New York, NY).

Plasmodium falciparum parasites.

Plasmodium falciparum parasites of strain 3D7 were maintained in culture, and gametocytogenesis was induced as described by Ifediba and Vanderberg (13). Aliquots (1 to 2 ml) of cultures containing parasites at the indicated stage of gametocytogenesis were transferred to the inner wells of a 24-well plate, and either control or test compounds were added. The outer wells were filled with sterile phosphate-buffered saline to decrease evaporation, and the plate was placed in a sealed container that was filled with blood gas (90% N2, 5% CO2 and 5% O2). The cultures were maintained at 37°C and fed daily with RPMI 1640 (no. 31800-022l; Invitrogen, Grand Island, NY) supplemented with hypoxanthine (10 mg liter−1) and 10% human serum. Parasitemia was monitored by the microscopic analysis of Giemsa-stained culture smears.

Cytotoxicity assays.

Cells from a mouse fibroblast line, NIH-3T3 (ATCC, Manassas, VA), or a human alveolar basal epithelial cell line, A549 (ATCC) (1 × 104 to 5 × 104 cells per well in a 96-well plate), were incubated with serial dilutions of epoxomicin (32 to 2,000 nM) or carrier (DMSO) alone in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. The concentration of DMSO was kept below 1%, which did not affect cell viability. Forty-eight hours after the addition of drug, cell viability was assayed using the Cell Titer 96 aqueous nonradioactive cell proliferation assay (Promega, Madison, WI) according to the manufacturer's instructions (http://www.promega.com/enotes/applications/0004/ap0017.htm). The assay uses a soluble tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), that is reduced by viable cells to fromazan, which can be measured by absorbance at 492 nM.

Plasmodium falciparum gametogenesis and mosquito feed assay.

Cultures of P. falciparum parasites (strain 3D7) containing mature gametocytes were incubated for 1 h in the presence of ALLN, epoxomicin, or DMSO alone. To assay gametogenesis and exflagellation, an aliquot (0.5 ml) of the test cultures was pelleted and resuspended in emergence medium (10 μM xanthurenic acid, 1.67 mg ml−1 glucose, 8 mg ml−1 NaCl, 8 mM Tris-Cl [pH 8.2]). After a 10-min incubation at room temperature, parasite morphology and the number of exflagellating males was evaluated at a ×400 magnification. To evaluate oocyst production, an aliquot (1.2 ml) of the test cultures was pelleted onto 125-μl packed erythrocytes. The supernatant was replaced with 120 μl of normal human serum containing active complement, mixed, and introduced into a water-jacketed membrane feeder maintained at 37°C (18). Anopheles stephensi (SxK Nij.) mosquitoes were allowed to gorge for 10 min and then were grown for 7 more days at 25 ± 1°C and 80% ± 10% humidity. The midguts then were dissected and stained in 1.0% mercurochrome to visualize the P. falciparum oocysts.

RESULTS

Cysteine protease inhibition.

Plasmodium falciparum cultures containing both asexual parasites and gametocytes were incubated with an inhibitor of serine and cysteine proteases (TPCK), cysteine protease inhibitors (E64d, ALLM, MLHF, or ALLN), or a carrier control (DMSO). A 24-h incubation with TPCK, even at a 100 μM concentration, had no effect on asexual parasites or gametocytes. Two days after drug treatment, the number of gametocytes per 1,000 red blood cells (RBCs) was 21% ± 0.09% (average ± standard deviation) for TPCK and 26% ± 0.3% for DMSO; therefore, this compound was not evaluated further. As previously reported for E64d, both it as well as ALLM caused a reduction in asexual-stage parasites (Fig. (Fig.1A)1A) as well as a pronounced enlargement of the food vacuole in asexual trophozoite-stage parasites, consistent with a block in hemoglobin digestion (Fig. (Fig.1B).1B). Following a 24-h treatment with E64d (100 μM) or ALLM (100 μM), sexual-stage parasitemia was significantly reduced (Fig. (Fig.1C),1C), and the majority of the stage II gametocytes also contained multiple enlarged food vacuoles (Fig. (Fig.1D).1D). Enlarged vacuoles were not observed in most parasites that had reached stage III prior to cysteine protease inhibitor treatment. Furthermore, once the drug was removed, the gametocytes continued to develop into morphologically normal stage III to V gametocytes (Fig. (Fig.1D).1D). In the experiment shown in Fig. Fig.2,2, ,22 days after E64d treatment there were 47 gametocytes/1,000 RBCs; 72% were stage II (47% vacuolated and 25% normal), 18% stage III (8% vacuolated and 10% normal), and 10% stage IV (2% vacuolated and 8% normal). In the control DMSO-treated culture, there were 42 gametocytes/1,000 RBCs; 64% were normal stage II gametocytes, and 36% were normal stage III gametocytes. Three days later (5 days after E64d treatment), there were 36 gametocytes/1,000 RBCs, no stage II gametocytes, 22% stage III gametocytes (14% vacuolated and 8% normal), 68% stage IV gametocytes (3% vacuolated and 65% normal), and 10% stage V gametocytes (5% vacuolated and 5% normal). In the control DMSO-treated culture, there were 63 gametocytes/1,000 RBC, 3% normal stage II gametocytes, 20% normal stage III gametocytes, 74% normal stage IV gametocytes, and 3% normal stage V gametocytes. These data demonstrate that cysteine protease inhibitors have access to the food vacuole and can successfully block hemoglobin digestion in gametocytes, but even at 100 μM concentrations the inhibitors do not have long-term effects. Once the inhibitor was removed, hemoglobin digestion and sexual differentiation resumed.

FIG. 1.
Effect of cysteine protease inhibitors on erythrocytic-stage parasites. P. falciparum cultures containing asexual (Asex) stages (A and B) or gametocytes (C and D) were incubated with MLHF (100 μM), E64d (100 μM), ALLM (100 μM), ...
FIG. 2.
Stage-specific effect of protease inhibitors on parasite morphology. Eight days after setup, P. falciparum gametocyte cultures were incubated with E64d (100 μM) or an equivalent amount of DMSO for 24 h. For the next 3 days, the cultures were fed ...

These findings are in marked contrast to the effect of another cysteine protease inhibitor, ALLN, at 100 μM (Fig. (Fig.1).1). After a 24-h incubation in ALLN (100 μM), only residual asexual stages are seen (Fig. (Fig.1A1A and B), and the remaining gametocytes are very thin and continue to shrink during the next 2 days, even in the absence of drug (Fig. (Fig.1C1C and D). Interestingly, a 10-fold lower concentration of ALLN (10 μM) resulted in the same phenotype as that of E64d and ALLM (Fig. (Fig.1),1), suggesting that at high concentrations ALLN has additional inhibitory effects. ALLN also has been reported to block proteasome activity; therefore, to evaluate whether the proteasome is essential for gametocyte maturation, a proteasome-specific inhibitor, epoxomicin, was tested (16, 27).

Proteasome inhibition.

Mixed asexual and gametocyte cultures (5.9 ± 1.7 stage II gametocytes/1,000 RBC, 20.8 ± 8.22 stage III gametocytes/1,000 RBC, and 1.78 ± 2.5 stage IV gametocytes/1,000 RBC) were incubated with serial dilutions of epoxomicin (10 nM to 1 μM) or DMSO as a carrier control for 24 h. The total gametocytemia following drug treatment is plotted as a percentage of the control DMSO-treated gametocytemia in Fig. Fig.3A,3A, and representative images of the parasites are shown in Fig. Fig.3B.3B. The parasites then were maintained in culture without drug for 2 more days (a total of 72 h after drug addition), and development was monitored by Giemsa-stained smears. During this time period gametocytes in the control, DMSO-treated culture had advanced to 8.7 ± 0.23 stage III gametocytes/1,000 RBC, 11.8 ± 0.08 stage IV gametocytes/1,000 RBC, and 2.9 ± 1.3 stage V gametocytes/1,000 RBC. In contrast, no morphologically normal parasites remained in the epoxomicin-treated cultures 72 h after treatment even at 10 nM, the lowest concentration tested.

FIG. 3.
Effect of proteasome inhibitors on intraerythrocytic asexual (Asex) and sexual stages (G'cyte). P. falciparum cultures containing both asexual- and sexual-stage parasites were incubated with 10 nM, 100 nM, or 1 μM epoxomicin or the equivalent ...

To compare the effect of epoxomicin on asexual and sexual parasites, the 24-h time point was used to calculate the 50% inhibitory concentrations (IC50s) (Fig. (Fig.3C).3C). Twenty-four hours after treatment, both ring-stage asexual parasites and gametocytes where observed in the 10 nM epoxomicin-treated cultures, but in the 100 nM and 1 μM epoxomicin-treated cultures the number of gametocytes and asexual-stage parasites decreased by 77 and 86%, respectively. In this experiment, the 24-h IC50s for gametocytes and asexual-stage parasites were 54 and 41 nM epoxomicin, respectively, while human (A549) and mouse (3T3) cell lines remained 80% viable in 1 μM epoxomicin. Similar inhibitory profiles for asexual- and sexual-stage parasites is very unusual for antimalarial drugs. The mature gametocyte IC50s reported for pyrimethamine, chloroquine, quinine, and even artemisinin are at least an order of magnitude higher than those for asexual stages, and when they are used clinically, these drugs do not eliminate gametocytes (5, 24). We confirmed this in our in vitro system by incubating a mixed asexual and stage V gametocyte culture with quinine, artemisinin, epoxomicin, or the appropriate carrier control. Quinine and artemisinin effectively killed asexual-stage parasites but had no effect on gametocytes, while only thin degrading gametocytes remained after epoxomicin treatment (Fig. (Fig.4A).4A). Images of parasites before and after treatment are shown under the graph (Fig. (Fig.4B4B).

FIG. 4.
Differential inhibition of asexual (Asex) parasites and mature stage V gametocytes. P. falciparum cultures containing asexual parasites and stage V gametocytes were treated for 24 h with quinine (10 and 1 μM), artemisinin (1 and 0.1 μM), ...

Malaria transmission-blocking activity.

The transmission-blocking potential of epoxomicin and ALLN was evaluated by testing both gametogenesis and oocyst production. Neither epoxomicin, ALLN, nor DMSO treatment blocked the ability of mature stage V gametocytes to undergo gametogenesis or exflagellation when stimulated with emergence media and cooled to room temperature, conditions that simulate the transition to the mosquito midgut. The average number of exflagellation centers per ×40 field for DMSO-treated gametocytes was 3.25, for ALLN (100 μM) was 2.75, and for epoxomicin (1 and 10 μM) was 2.75 and 4.33, respectively. Epoxomicin also did not block the processing of the gamete surface antigen Pfs230 during RBC emergence (Fig. (Fig.5).5). In contrast, ALLN did effectively block Pfs230 processing, as expected from our previous work using the cysteine protease inhibitor E64d (10). The lack of effect of epoxomicin on Pfs230 processing is consistent with its reported specificity for the proteasome, not cysteine proteases. These data also suggest that the gametocytocidal activity of both epoxomicin and ALLN are due to proteasome, not cysteine protease, inhibition. In marked contrast to the gametogenesis and exflagellation results, further development into oocysts was significantly reduced when either epoxomicin- or ALLN-treated parasites were fed to Anopheles mosquitoes (Table (Table1).1). In total, 341 oocysts were produced in the 70 mosquitoes fed with DMSO-treated parasites, while no oocysts were produced by epoxomicin-treated parasites and only a total of 3 oocysts were produced in the 43 mosquitoes fed ALLN-treated parasites. This is similar to the significant block found with E64d and suggests that both the cysteine proteases and the proteasome play important roles in sporogonic development (10).

FIG. 5.
Proteolytic processing of the gamete surface antigen Pfs230 during gametogenesis. P. falciparum cultures containing stage V gametocytes were incubated with ALLN (A) (100 μM), epoxomicin (E) (1 and 10 μM), or the equivalent amount of DMSO ...
TABLE 1.
P. falciparum oocyst productiona

DISCUSSION

The work reported here demonstrates that nanomolar concentrations of the proteasome inhibitor epoxomicin effectively kill parasites throughout the asexual, sexual, and mosquito midgut stages of the life cycle, but it did not reduce the viability of mouse or human cell lines (3T3 or A549, respectively). These findings provide support for the further development of this class of compounds as antimalarial agents with the potential to prevent transmission in addition to reducing clinical symptoms caused by asexual parasitemia. In contrast, even micromolar concentrations of artemisinin and quinine did not affect mature gametocytes, and although cysteine protease inhibitors effectively inhibited hemoglobin digestion in asexual parasites and early gametocytes, they had no effect on gametocytes after stage II. Moreover, once the cysteine protease inhibitor is removed, sexual differentiation resumes. This suggests that although these compounds effectively inhibit the growth of asexual parasites, sexual development is not blocked and consequently malaria transmission continues impeding efforts to eradicate malaria.

Morphologically, gametocytes treated with proteasome inhibitors begin to decrease in width from both ends, but length is not affected, resulting in a thin needle-like parasite. This suggests that the cytoplasmic contents are degraded before the plasma membrane and underlying microtubules. Compared with the 10- to 12-day life span of the gametocyte, the degradation occurs rather rapidly (1 to 2 days), suggesting that the lack of proteasome activity has a toxic effect and does not just block growth or development. Proteasome inhibition is known to disrupt the degradation of intracellular proteins, including cell cycle regulators (11). In rapidly proliferating tumor cells, this can lead to cell cycle arrest and then apoptosis, but quiescent cells often are not affected (6). The differential effect of proteasome inhibitors on proliferating and quiescent cells led to the development of bortezomib (Velcade or MLN0341; Millennium Pharmaceuticals, Inc.), a reversible boronic acid dipeptide proteasome inhibitor that is currently in human trials as a treatment for malignant melanoma. Analogs of epoxomicin with epoxyketone active sites also are being screened for therapeutic efficacy against tumor cells (8).

The core proteasome is made up of four rings of seven polypeptides each (6). The two outer rings are identical, and the subunits are designated alpha 1 to 7. The two inner rings also are identical, and the subunits are designated beta 1 to 7. Beta subunits 1, 2, and 5 are the active proteases, and all three have an N-terminal threonine that is the target of most of the proteasome inhibitors identified to date. All subunits of the proteasome, as well as an ortholog of the bacterial threonine protease ClpQ/hslV, PfhslV (PFL1465c), have been identified in the P. falciparum genome and are expressed through gametogenesis (12). The interaction of epoxomicin with the beta 5 and 2 subunits in P. falciparum was demonstrated by its ability to competitively block the labeling of the beta 5 and beta 2 subunits by biotinylated adamantineacetyl-lysinyl(biotinyl)-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulfone (17). The P. falciparum beta 2 (PF13_0156) and 5 (PF10_0111) subunits are 55 and 53% identical to the corresponding human orthologs, respectively (1, 2). The active-site residues are well conserved, but the amino acids surrounding the central peptide channel are distinct, suggesting that analogs can be developed that preferentially bind the Plasmodium enzymes (6). The further evaluation of the potent gametocytocidal activity of epoxomicin also may provide new insights into the genes required for parasite survival and lead to the development of additional antimalarials that have a dual role in inhibiting clinical disease and transmission.

Acknowledgments

This investigation received financial support from Public Health Service grants AI40592 and AI48826 from the National Institute of Allergy and Infectious Diseases and from the James A. and Marion C. Grant Research Fund.

We thank M. Bogyo, Stanford University, for expert advice and A. Suri and U. Hasan, Loyola University Chicago, for technical assistance.

Footnotes

[down-pointing small open triangle]Published ahead of print on 3 August 2009.

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