The clones 3D7, HB3, Dd2, and W2 of P. falciparum used in this study were obtained from the Malaria Research and Reference Reagent Resource Center (MR4).

Parasites were cultured by the method of Trager and Jensen 16 by using a gas mixture of 3% O₂, 3% CO₂, and 94% N₂. RPMI medium 1640 was supplemented with 30 mg/liter hypoxanthine (Sigma), 25 mM Hepes (Sigma), 0.225% NaHCO₃ (Sigma), 0.5% Albumax I (Life Technologies, Grand Island, NY), and 10 μg/ml gentamycin (Life Technologies). Bortezomib was purchased from the University of Connecticut Health Center Pharmacy. ZL₃B was purchased from Boston Biochemical Inc. (Cat# I-120). Parasite synchronization was obtained with three successive 5% sorbitol treatments 17. To determine visually the stage of the parasite life cycle, fixed smears of the P. falciparum-infected erythrocytes were stained with Giemsa stain and analyzed by bright-field microscopy.

The susceptibility of parasites to different compounds was assessed by tritiated hypoxanthine uptake as described by Desjardins and colleagues 18. Briefly, infected erythrocytes (2% hematocrit, 3% rings) were washed and incubated with the appropriate drugs at the listed concentrations in hypoxanthine-free media for 48 hours. 200 μL of the mixture was then added to a 96 well plate with ³H-hypoxanthine at a concentration of 0.5 μCi/well. Following an incubation of 24 hours, the cells were washed on an ultrafilter and radioactivity was counted using a scintillation counter. IC₅₀'s are represented in nM. Values are means ± standard deviation of three independent experiments each performed in triplicate. These experiments were performed at least three times with similar results.

The cell permeable peptide boronate Z-Leu-Leu-Leu-B(OH)₂ (ZL₃B) (Fig. 1A) is a specific and potent proteasome inhibitor that blocks the growth of the bloodstream form of the protozoan parasite Trypanosoma brucei with a 50% inhibitory concentration (IC₅₀) of 0.32 nM in culture 14. In order to examine the antimalarial activity of ZL₃B, we have tested the effect of increasing concentrations of this compound up to 200 nM on the intraerythrocytic life cycle of P. falciparum in culture by following the incorporation of radiolabeled hypoxanthine into parasite nucleic acids. The study was performed with four different strains of P. falciparum (3D7, HB3, W2 and Dd2) that are either sensitive or have different levels of resistance to the antimalarial drugs pyrimethamine and chloroquine (Fig. 1C–F and Table 1). ZL₃B was found to inhibit parasite proliferation with an IC₅₀ values between 34 and 45 nM. Due to the strong antimalarial activity of ZL₃B, we speculated that the ZL₃B analog and clinically approved peptide boronate, bortezomib (Fig. 1B) might have a similar antimalarial activity. Bortezomib is a boronic acid dipeptide and a reversible inhibitor of the chymotrypsin-like activity of the 20S proteasome 15. It strongly and selectively inhibits the proteasome, has substantial cytotoxicity against a broad range of human tumor cells and has shown excellent anti-tumour activity in preclinical and clinical trials 15. Bortezomib was approved by the U.S. Food and Drug Administration and the European commission for the treatment of advanced multiple myeloma, and more recently, it received a fast track status for relapsed and refractory mantle cell lymphoma 13. We first analyzed the antimalarial activity of bortezomib by using the hypoxanthine assay and found that the drug inhibited proliferation of P. falciparum 3D7, HB3, W2 and Dd2 strains with IC₅₀ values ranging between 31 and 43 nM (Fig. 1D and Table 1). As a control, we confirmed the IC₅₀ of chloroquine and pyrimethamine in the four strains (Fig. 1E and 1F, and Table 1). As expected, strain 3D7 was sensitive to both compounds with IC₅₀ of 6 ± 0.2 and 5 ± 1.1 nM, respectively; strain HB3 was sensitive to chloroquine (IC₅₀: 8 ± 1.4 nM) and resistant to pyrimethamine (IC₅₀: 500 ± 45 nM); strain W2 was moderately resistant to chloroquine (IC₅₀: 90 ± 3.7 nM) and highly resistant to pyrimethamine (IC₅₀: 1.5 ± 0.006 μM), and Dd2 was highly resistant to both chloroquine (IC₅₀: 300 ± 21 nM) and pyrimethamine (IC₅₀: 2.5 ± 0.097 μM). To further confirm the inhibitory effects of bortezomib and ZL₃B on P. falciparum growth, we employed the pLDH colorimetric assay, which measures the production of parasite specific lactate dehydrogenase activity 192021. Consistent with the results of the hypoxanthine incorporation assay, both compounds were found to inhibit equally well chloroquine- and pyrimenthamine- sensitive and resistant strains (not shown). Noteworthy, these studies were also consistent with the finding that another boronic derivative, MLN-273, inhibits the intraerythrocytic development of P. falciparum 12.

To determine the developmental stage during which bortezomib and ZL₃B exert their antimalarial effects, P. falciparum cultures were synchronized and the proteasome inhibitors were added to the culture medium at different times following parasite invasion and a final concentration of 100 nM. Culture samples were collected every 6 hours and parasite intraerythrocytic developmental progression was monitored by Giemsa staining and light microscopic analysis (Fig. 2A). As a control, an untreated culture of P. falciparum-infected erythrocytes was monitored. In the absence of bortezomib or ZL₃B, the parasite displayed a normal cycle progression from rings to trophozoites, trophozoites to schizonts and schizonts to rings in approximately 44 hours (Fig. 2A). Addition of bortezomib or ZL₃B during the ring (8, 16 h post-invasion) or early trophozoite (24 h post-invasion) stages resulted in a complete blockage of developmental progression and subsequent death of the parasites. Treatment with these compounds during the late trophozoite stage (32 h post-invasion) only partially blocked parasite progression (Fig. 2B). On the other hand, treatment during the schizont stage (40 h post-invasion) had no effect on parasite progression (Fig. 2A). This stage-specific inhibitory effect of bortezomib and ZL₃B was quantified by counting the number of rings that developed 48 hours post-invasion in the absence or presence of the compounds. Consistent with the previous analysis, no rings could be detected from cultures treated with bortezomib or ZL₃B 8, 16 or 24 h post-invasion. In contrast, treatment with these compounds 32 h post-invasion reduced the number of rings in comparison to untreated parasites by 40% only (Fig. 2B) and treatment 40 h post-invasion was completely ineffective (not shown). Together these data suggest that these compounds inhibit parasite development and multiplication and have a lesser effect on the release of merozoites from the infected erythrocytes or on the invasion of new red blood cells by the released merozoites. Although a direct effect of these compounds on the proteasome has not been investigated, our data along with the known mode of action of these compounds in other cell lines suggest an important role for the P. falciparum proteasome in parasite development and DNA synthesis.

The recommended adult dose of bortezomib for treatment of myeloma is 1.3 mg/m²; and in children the compound is used at a dose of 1.2 mg/m² 222324. The mean peak plasma concentration (Cmax) determined 5 min after drug administration at doses between 1.3 and 1.7 mg/m² was 63 ± 16 ng/ml and the mean area under the concentration-time curve extrapolated to infinity (AUCinf) was 27 h ng/ml 23. These values are 2 to 4-fold the IC₅₀ observed with bortezomib in P. falciparum. In vivo studies to determine the dose and tolerability of this compound for treatment of malaria are warranted.