PROJECT SUMMARY Malaria, which affected 212 million people worldwide in 2015, is caused by five parasite species, the most virulent of which is Plasmodium falciparum. The World Health Organization recommends artemisinin (ART)- based combination therapies (ACTs), which consist of a potent but short-lived artemisinin derivative and a longer-lived partner drug, for treatment of non-complicated falciparum malaria. Alarmingly, resistance to ART has arisen in the last decade in Southeast Asia. To exacerbate matters, resistance to the ACT partner drug piperaquine is now arising in Southeast Asia. A few cases of ART treatment failure have been documented in Africa, but there is no widespread resistance in this region. The search for new combination therapies is urgently needed. A combination of dihydroartemisinin (DHA), the active metabolite of ART, and the P. falciparum-specific proteasome inhibitor WLW demonstrates synergy when exposed to early ring stages of ART-resistant (ARTR) parasites. We have extended these findings, demonstrating synergy at the trophozoite stages. We have also identified novel synergistic interactions with WLW and the endoperoxide OZ439, the endoplasmic reticulum-associated degradation (ERAD) inhibitor EERSTI, and the deubiquitinase (DUB) inhibitor b-AP15. The goal of this proposal is to understand the molecular mechanisms and pathways underlying these synergistic interactions to inform combination therapies going forward. We hypothesize that proteasome inhibitors synergize with drugs including artemisinins that cause an imbalance between the load of proteins to be degraded and the protein degradation capacity, leading to increased stress, a terminal UPR, and eventual death. We will examine ubiquitin levels, proteasome activity, and cellular stress responses in dissecting the molecular reasons underlying synergy with proteasome inhibitors. The answers illuminated by these experiments will allow us to rationally target parasite proteins and/or pathways for future drug combination therapies.