In view of increasing levels of P. falciparum resistance to antimalarial drugs currently in use, there is an urgent need to develop new treatment strategies. Proteasome inhibitors are in the focus of drug development for various diseases and candidates have already entered clinical trials . In plasmodia several studies show the potential of a single proteasome inhibitor as a drug development candidate [2–5]. Even though different growth detection methods give highly comparable results, assay parameters, mainly the initial parasitaemia used in the drug susceptibility assay, affects IC50 values . Thus comparison of inhibitor activities between different studies is problematic, particularly if no comparator drug is tested in parallel. Here, the first comprehensive study on proteasome inhibitors against P. falciparum is provided. The most promising inhibitor amongst all classes tested was identified and its activities were compared with artesunate, one of the most active antimalarial compounds known. Moreover, differences in activities between the inhibitor classes are not biased by assay parameters because all compounds were tested in parallel and under exactly the same assay conditions.
The knowledge of activity of antimalarial drug candidates against field isolates is critical to estimate their potential in the drug development process. Freshly isolated parasites are heterogeneous in their genetic background and represent the local plasmodial population, including pre-existing mutations that account for drug resistance, whereas laboratory isolates are oligo- or monoclonal and are heavily selected for in vitro growth. It is important to establish the variability of efficacy of antimalarials (as it is seen in the range of IC50 values) against parasites with a diverse genetic background. The discrepancies seen between assays carried out on (fresh) patient isolates and on laboratory adapted strains cannot be predicted. Bortezomib showed a remarkably lower activity against P. falciparum field isolates then it was seen in laboratory strains. Furthermore, the variability in susceptibilities of patient isolates to one proteasome inhibitor class can be compared to the variability of susceptibilities to another compound. These comparisons can point to potential mechanisms of drug action as well as drug resistance. Resistance against chloroquine is highly prevalent in the study area and exclusion of cross-resistance with chloroquine is of major concern.
Peptide a',β'-epoxyketones are the most potent and specific proteasomal inhibitor class in mammalian cells so far . In P. falciparum the peptide epoxyketone epoxomicin, an actinomycetes metabolite, showed the highest antimalarial activity in laboratory strains as well as in field isolates, independent of the chloroquine-susceptibility of the parasites. The narrow range of activity in the individual samples argues for a low potential of resistance-development. Interestingly, caspase-like subunit specific YU102  did not affect parasite growth, whereas YU101 , a chymotrypsin-like specific inhibitor, inhibited parasites in the low nanomolar range. YU101 and YU102 are synthetic derivates and their peptide modifications were designed to target only one proteasomal proteolytic subunit with a high degree of specificity and potency. Even though epoxomicin is not specific for the plasmodial proteasome, YU-compounds show that it is principally possible to design epoxyketones towards subunit specific inhibition. If more is known about plasmodial proteasome particularities, like their low complexity regions in their active subunits , engineering of plasmodia specific epoxyketones should be feasible. The peptide vinyl sulfone analogue of MG132, Z-L3-VS, was considerable more potent in P. falciparum then Ada-Ahx3-L3-VS, a vinyl sulfone with an amino-terminal extension. This was surprising because in mammalian proteasomes the extended peptide vinyl sulfones were shown to be superior in potency over their shorter counterparts . Presumably, the amino-terminal extension of Ada-Ahx3-L3-VS is hampering the molecule's permeability to the three membranes of P. falciparum infected erythrocytes. Although MG132 showed promising antimalarial activity regardless of the chloroquine susceptibility of the parasites, a major drawback of peptide aldehydes is their lack of proteasomal specificity. The proline and arginine-rich peptides PR39 and PR11 regulate the proteasome activity in a substrate-specific manner. They are unique in that they bind allosterically to the α subunits of the proteasome and change the shape of the proteasome. Only the degradation of a small subset of proteins is affected and thus antimicrobial peptides are less toxic than conventional competitive proteasome inhibitors . In plasmodia no activity could be observed, however, inhibition of purified plasmodial proteasomes was not investigated. Although lactacystin and gliotoxin have already been shown active against P. falciparum [3, 5], in the light of other proteasome inhibitor classes and comparator drugs their activity was of minor significance.
Currently, proteasome inhibitors are intensely investigated for their antimicrobial activity by several academic groups and companies. We hope to set a standard for pre-clinical in vitro studies of antimalarial inhibitors and look forward to the further development of lead compounds into clinical trials.