Comprehensive study of proteasome inhibitors against Plasmodium falciparum laboratory strains and field isolates from Gabon

Abstract

Background: The emergence and spread of Plasmodium falciparum resistance to almost all available antimalarial drugs necessitates the search for new chemotherapeutic compounds. The ubiquitin/proteasome system plays a major role in overall protein turnover, especially in fast dividing eukaryotic cells including plasmodia. Previous studies show that the 20S proteasome is expressed and catalytically active in plasmodia and treatment with proteasome inhibitors arrests parasite growth. This is the first comprehensive screening of proteasome inhibitors with different chemical modes of action against laboratory strains of P. falciparum. Subsequently, a selection of inhibitors was tested in field isolates from Lambaréné, Gabon.

Methods: Epoxomicin, YU101, YU102, MG132, MG115, Z-L(3)-VS, Ada-Ahx(3)-L(3)-VS, lactacystin, bortezomib (Velcade), gliotoxin, PR11 and PR39 were tested and compared to chloroquine- and artesunate-activities in a standardized in vitro drug susceptibility assay against P. falciparum laboratory strains 3D7, D10 and Dd2. Freshly obtained field isolates from Lambaréné, Gabon, were used to measure the activity of chloroquine, artesunate, epoxomicin, MG132, lactacystin and bortezomib. Parasite growth was detected through histidine-rich protein 2 (HRP2) production. Raw data were fitted by a four-parameter logistic model and individual inhibitory concentrations (50%, 90%, and 99%) were calculated.

Results: Amongst all proteasome inhibitors tested, epoxomicin showed the highest activity in chloroquine-susceptible (IC50: 6.8 nM [3D7], 1.7 nM [D10]) and in chloroquine-resistant laboratory strains (IC50: 10.4 nM [Dd2]) as well as in field isolates (IC50: 8.5 nM). The comparator drug artesunate was even more active (IC50: 1.0 nM), whereas all strains were chloroquine-resistant (IC50: 113 nM).

Conclusion: The peptide alpha',beta'-epoxyketone epoxomicin is highly active against P. falciparum regardless the grade of the parasite's chloroquine susceptibility. Therefore, inhibition of the proteasome is a highly promising strategy to develop new antimalarials. Epoxomicin can serve as a standard to compare new inhibitors with species-specific activity.

Figure 1

Chemical structure of proteasome inhibitors. Pharmacophores are shown in red. PR39 and PR11: amino acid sequence is shown; binding sites are so far unknown.

Figure 2

Distribution of individual 50% inhibitory concentrations. Distribution of single IC50s of artesunate (A), chloroquine (CQ), epoxomicin (Ep), MG132 (MG), bortezomib (B) and lactacystin (L) in P. falciparum field isolates. Outlier box plots show median IC50 and ends of the box are the 25% and 75% quantiles, respectively. The whiskers extend from the ends of the box to the outermost data point that falls within the distances computed. Dots outside whiskers are possible outlier IC50s. Note: concentration (nM) is shown in logarithmic scale.