Figure 1.
Morphologies of inhibitor-treated parasites.
Parasites were cultured without (control) or with indicated inhibitors (21.7 µM E64, 0.024 µM epoxomicin, 0.812 µM lactacystin, 0.133 µM MG132, and 220.6 µM pepstatin; concentrations are nearly 3 times the IC50) for 48 hours, and the morphologies of parasites on Giemsa-stained smears were evaluated at indicated time points. Note that both E64- and MG132-treated parasites have enlarged food vacuoles occupying almost the entire parasite, whereas parasites treated with epoxomicin, lactacystin, and pepstatin are pyknotic.
Figure 2.
Accumulation of undegraded hemoglobin and ubiquitinated proteins in inhibitor-treated parasites.
Cultures containing equal number of early trophozoite stage parasites were grown in the presence of 0.1% DMSO (control) or indicated inhibitors (21.7 µM E64, 0.024 µM epoxomicin, and 0.133 µM MG132; all at concentrations nearly 3 times the IC50) for 10 hours. Parasites were lysed and equal amounts of supernatants were used to assess accumulation of undegraded hemoglobin (A) and ubiquitinated proteins (B) as described in the Materials and Methods section. A. Coomassie-stained SDS-PAGE gel, showing significantly more amount of undegraded hemoglobin (marked with arrow) in parasites treated with E64 and MG132 than control and epoxomicin-treated parasites. B. Western blot using anti-ubiquitin antibodies showed markedly more intense high molecular weight signal in the MG132 and epoxomicin samples than in control and E64 samples, which is indicative of accumulation of ubiquitinated proteins in the MG132 and epoxomicin samples. The experiment was repeated twice, samples from each experiment were analyzed three times for A and two times for B, and the results were reproducible. M, molecular weight in kD; Hb, hemoglobin.
Figure 3.
Effect of inhibitor washout on parasite growth.
Ring stage parasites were cultured in the presence of DMSO (control) or chloroquine or indicated inhibitors (21.7 µM E64, 0.024 µM epoxomicin, 0.133 µM MG132; concentrations are nearly 3 times the IC50) for one cycle, and then without any treatment for next two cycles as described in the Materials and Methods section. Parasite growth was measured at the end of each cycle using the SYBR Green-1 dye. The results with standard deviation error bars from three independent experiments, each in triplicate, are shown as arbitrary fluorescence units of the dye. Similar fluorescence values of parasites treated with MG132, epoxomicin, and chloroquine indicate that MG132 and epoxomicin, like chloroquine, irreversible block parasite development.
Figure 4.
Inhibition of cysteine protease and proteasome activities by MG132.
A. Inhibition of total soluble extracts of P. falciparum. Extracts of trophozoite/schizont stage parasites were prepared by freeze-thaw lysis and ultrasonic treatment of cells. Identical aliquots were treated with 1.25% DMSO (control), 10 µM E64, 1 µM epoxomicin (Epox), 1 µM MG132, or the indicated inhibitor combinations (10 µM E64 + 1 µM epoxomicin, 10 µM E64 + 1 µM MG132, or 1 µM epoxomicin + 1 µM MG132) for 10 min at 37°C, and protease activities were determined by monitoring hydrolysis of fluorogenic peptide substrates for 30 min at 37°C as described in Materials and Methods. The substrates used were Z-LR-AMC (LR) for cysteine protease activity, Suc-LLVY-AMC (LLVY) for chymotrypsin-like proteasome activity, and Ac-RLR-AMC (RLR) for trypsin-like proteasome activity. Activities of inhibitor-containing reactions were compared with those of controls and expressed as percent inhibition. The results shown are means of two independent experiments, each performed in duplicate. B. Inhibition of recombinant falcipains. Recombinant FP2 (5 nM) or FP3 (10 nM) was incubated with 1% DMSO (control) or indicated inhibitors (all 10 µM) in sodium acetate assay buffer for 30 min at room temperature. Z-LR-AMC (25 µM) was added to the reaction, and enzyme activity was measured by monitoring substrate hydrolysis for 30 min at 37°C. Enzyme activities of inhibitor-containing reactions were compared with those of controls and expressed as the percent inhibition. The results shown are means of three independent experiments, each performed in triplicate.
Figure 5.
Antimalarial effects of combinations of proteasome and falcipain inhibitors.
Parasites were cultured in the presence of varied combinations of the indicated inhibitor, and the 50% inhibitory concentration of each combination was determined to calculate fractional inhibitory concentrations (FIC). FICs from three independent experiments, each carried out in duplicate were used to construct isobolograms as described in Materials and Methods section. Mean FICs (ΣFIC) 0.5-1.0, <0.5, and >1.5 are indicative of additive, synergistic and antagonistic interactions, respectively. A. The isobolograms indicate additive interactions for the indicated combinations.
Figure 6.
The isobolograms indicate additive, synergistic, and antagonistic antiparasitic effects of E64-pepstatin, MG132-pepstatin, and epoxomycin-pepstatin combinations, respectively.