Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes

Abstract

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

Figure 1

Activity-based probe BMV037 as a tool to monitor activity of multiple β subunits of the Plasmodium proteasome. (A) Structure of BMV037. The epoxyketone electrophile is shown in red and the Cy5 fluorophore shown in blue. (B) Time dependent labeling of the purified P. falciparum 20S proteasome and schizont lysates by BMV037. The location of the β subunits is based on mass spectrometry results shown below. (C and D) Mass spectrometry analysis of labeled subunits of the P. falciparum 20S proteasome. Purified proteasome (20 nM) was incubated with 10 μM of BMV037 for 3 h, and analyzed by SDS PAGE. Left panel shows coomassie stain, and right panel shows the same gel under fluorescent scan. The gel was divided into 14 slices and protein content of each slice was analyzed by mass spectrometry. Graph of spectral counts of individual catalytic subunits: β1 (Unitprot: Q8I0U7), β2 (Uniprot: Q8I6T3), and β5 (Uniprot: Q8IJT1) in each gel slices is shown.

Figure 2

Subunit selective mammalian proteasome inhibitors do not retain subunit selectivity in P. falciparum. (A) Structures of the proteasome inhibitors. Intact human foreskin fibroblasts (HFF; B) or intact P. falciparum schizonts (C) were treated for 1 h with each inhibitor at the indicated final concentrations at 37 °C followed by postlysis labeling with 10 μM BMV037 (for P. falciparum) or 2 μM MV151 (for HFF). Samples were analyzed by SDS-PAGE followed by scanning of the gels for fluorescence using a flatbed laser scanner. The position of each of the labeled active β subunits is indicated.

Figure 3

Selective inhibition of β5 subunit during schizogony corresponds to block in parasite replication. (A) Structure of PR709A. Inhibitor competition assay for purified proteasomes (top) and intact cells (bottom) are shown. All inhibitor treatment was performed for 1 h at 37 °C followed by inhibitor washout and labeling with 10 μM BMV037 (P. falciparum) or 2 μM MV151 (HFF). (B) Quantification of residual proteasome β subunit activities (black, blue, and red lines) compared to parasite viability relative to control (schizont treatment; n = 6) upon treatment with increasing doses of PR709A. (C) Similar data as part B but for HFF cells treated with PR709A for 1 h. Cells were treated for 1 h with inhibitor, followed by a washout period of 72 h after which viability was determined (n = 3). Error bars represent standard error of the mean (SEM).

Figure 4

Selective inhibition of β2 does not affect parasite growth, but coinhibition of catalytic subunits at high concentration of LU102 blocks parasite replication at blood and liver stage. (A) Quantification of residual proteasome β subunit activity as measured by BMV037 labeling in parasites treated for 1 h with LU102 (red, blue, and black lines) compared to overall parasite viability as measured in parasite replications assays (green line). (B) Same results as in part A,except for intact HFF cells. Error bars represent standard error of the mean (SEM) from n = 3. (C) Synchronized P. falciparum culture of schizonts, trophozoites, or rings were pulsed for 1 h with 25 μM LU102 at 37 °C. For sporozoite treatment, Plasmodium berghei sporozoites freshly dissected from salivary glands of infected mosquitoes were treated for 30 min at 20 °C and then placed on HepG2 cells for invasion. Data is presented as the percent of parasites relative to the DMSO control. Error bars represent SEM from n = 3.

Figure 5

In vivo assessment of subunit selective inhibitors in Plasmodium chabaudi mouse model of malaria. (A) Infected Balb/c mice were treated with 50 mg/kg of PR709A (n = 5) or vehicle (n = 4) for 3 consecutive days via tail vein injections starting from day 2 postinfection. Treatment was administered at night. (B) P. chabaudi infected Balb/c mice were treated with 20 mg/kg of LU102 (n = 4) or vehicle (n = 4) for 4 consecutive days via i.v. injections. (C) Infected mice were treated with vehicle or combination of LU102 and PR709A (n = 4 for each group), at the indicated amounts. Compounds were administered at night. The dosing schedule is indicated by the dots below the day. Percent parasitemia was measured by blood smear counts and FACS analysis. (D) Plot of data from panels A–C expressed as a percentage of the vehicle treated mice (Color of each bar corresponds to the same color used in A–C). Error bars represent SEM.