Covalent Plasmodium falciparum-selective proteasome inhibitors exhibit a low propensity for generating resistance in vitro and synergize with multiple antimalarial agents.

Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the beta 2, beta 5 or beta 6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria. Author summary The spread of artemisinin-resistant Plasmodium falciparum malaria across Southeast Asia creates an imperative to develop new treatment options with compounds that are not susceptible to existing mechanisms of antimalarial drug resistance. Recent work has identified the P. falciparum proteasome as a promising drug target. Here, we report potent antimalarial activity of highly selective vinyl sulfone-conjugated peptide proteasome inhibitors, including against artemisinin-resistant P. falciparum early ring-stage parasites that are traditionally difficult to treat. Unlike many advanced antimalarial candidates, these covalent proteasome inhibitors do not readily select for resistance. Selection studies with cultured parasites reveal infrequent and minor decreases in susceptibility resulting from point mutations in components of the 26S proteasome, which we model using cryo-electron microscopy-based structural data. No parasites were observed to be cross-resistant to both compounds; in fact, partial resistance to one compound often created hypersensitivity to the other. We also document potent synergy between these covalent proteasome inhibitors and multiple classes of antimalarial agents, including dihydroartemisinin, the clinical candidate OZ439, and the parasite transmission-blocking agent methylene blue. Proteasome inhibitors have significant promise as components of novel combination therapies to treat multidrug-resistant malaria.