Plasmodium falciparum is responsible for most malaria-associated deaths, and emerging resistance to the frontline artemisinin (ART) drug family has emphasized the urgent need for novel antimalarial agents. Now, writing in Nature, Bogyo et al. report the identification of a peptidic inhibitor of the Plasmodium proteasome and show that it inhibits growth of ART-resistant parasites and clears infection in mouse malaria models.

Credit: Kiyoshi Takahase Segundo/Alamy Stock Photo

The Plasmodium proteasome represents a key target for antimalarial drugs owing to its essential role in protein homeostasis, which influences various cellular processes. However, although existing proteasome inhibitors are toxic for P. falciparum at all stages of its life cycle, they also cross-react with the mammalian proteasome. Bogyo et al. therefore set out to identify differences in the specificities of the human and the P. falciparum proteasomes and therapeutically exploit this information to develop a novel antimalarial agent.

The authors collaborated with O'Donoghue and Craik at the University of California, San Francisco, to determine the substrate preferences of the activated human and P. falciparum proteasomes by monitoring the proteosomal degradation pattern of 228 diverse synthetic tetradecapeptides using liquid chromatography tandem mass spectrometry. By generating a frequency profile indicating which amino acids were most- and least-favoured in the subsites surrounding each cleaved bond, they identified 113 and 157 sites that are uniquely cleaved by the P. falciparum and human proteasomes, respectively. Major differences occurred on the amino-terminal side of the cleavage site, with the P. falciparum proteasome displaying a strong preference for aromatic residues, specifically Trp, at the P1 and P3 sites.

Using this information, the authors next designed inhibitors based on the canonical tri-leucine scaffold found in common proteasome inhibitors, such as MG132. By systematically replacing Leu residues at the P1 and P3 positions with Trp, they generated the peptide vinyl sulfones LLW-vs, WLL-vs and WLW-vs. Interestingly, WLW-vs specifically inhibited the β2 subunit of the parasite proteasome, while WLL-vs inhibited both the parasite β2 and β5 subunits, leading to more potent parasite-killing activity.

Determination of the structure of the P. falciparum proteasome bound to the inhibitors, using high-resolution cryo-electron microscopy and single-particle analysis, revealed an unusually open active site on β2 that could accommodate the two Trp side chains of the inhibitor. This analysis also provided information about the active-site architecture and accessibility at the β2 and β5 sites.

The authors then investigated the antimalarial effects of their compounds using live P. falciparum cultures and found that proteasome inhibition corresponded with a decrease in parasite survival. Importantly, WLL-vs was 1,000-times more toxic towards parasite cells than primary human fibroblasts.

Similarly, in a rodent Plasmodium chabaudi infection model, a single bolus dose of WLL-vs administered by tail vein injection reduced parasite burden to nearly undetectable levels, without signs of toxicity.

Finally, the newly developed proteasome inhibitors showed potency against field isolates of ART-resistant parasites and were highly synergistic with dihydroarteminisin, suggesting their potential to enhance ART activity and reduce the emergence of resistance.

These findings demonstrate that the Plasmodium proteasome is sufficiently unique from the human proteasome for selective targeting and highlight a potential strategy for ART-sensitization. Optimization of WLL-vs to generate compounds with improved potency and oral bioavailability is currently ongoing.