Project Summary/Abstract In 2020, with over 250 million debilitating cases and over half a million deaths, mostly in young children, malaria is a persistent global health crisis. The malaria-causing parasite Plasmodium falciparum (Pf) has developed resistance to most antimalarial drug deployed, including the backbone artemisinins (ARTs). ART and its semi-synthetic analogs are considered essential for malaria treatment. ARTs are prodrugs that are activated within the parasites to form a reactive radical that covalently attacks proteins, lipids and other cellular constituents. ART resistance is widespread in Southeast Asia and has been reported in Africa. ART combination therapy (ACT) is a mainstay for treatment of malaria, but its efficacy can be derailed when a two-drug combination becomes de facto monotherapy. Moreover, extended exposure of Pf to ACTs induces multidrug tolerance. We recently showed that inhibitors specific for the Pf proteasome (Pf20S) kill Pf in each stage of its life cycle and synergize with ART, overcoming ART resistance. This proposal builds on our discovery that a covalent hybrid of an ART analogue and a Pf20S inhibitor that we call an artezomib (ATZ) can enhance ART action and overcome resistance to each of its components. We have synthesized ATZs that are more potent Pf20S inhibitors than their component Pf20S inhibitor. They not only kill wild type and ART-resistant (K13 mutant) Pf, Pf with proteasome mutations that confer resistance to the Pf20S inhibitor, but also kill Pf that expresses both ART-resistant and PI-resistant mutations. We propose the following mechanism by which ATZs overcome resistance to the Pf20S inhibitor within them: We found that upon activation of ATZ in the parasites, the ART component binds Pf proteins, like activated ART itself. The Pf ubiquitin proteasome system digests ATZ-bound proteins into oligopeptides, some of which display the Pf inhibitor component of the ATZ. We hypothesize that extended contact of ATZ-bearing peptides within the Pf20S active site augments the binding of the Pf20S inhibitor component of the ATZ, overcoming the decreased binding otherwise conferred by Pf20S point mutations. Thus, an ATZ can overcome resistance to each of its components. In mouse models of malaria, an ATZ drove P. berghei below the limit of detection and suppressed recrudescence of a P. berghei ART-resistant K13 mutant and doing so better than ART. In Aim 1 of this proposal, we will conduct lead optimization to improve ATZs' potency, selectivity and ATZs' pharmacokinetic properties. In Aim 2, we will explore ATZs' mechanism of action; attempt to select for ATZ-resistant parasites; determine the frequency and mechanism of resistance, if any; and study antimalarial activity of ATZs in stages of the Pf life cycle when ART alone is ineffective. Aim 3 will test the efficacy of ATZs in mice, including humanized mice infected with Pf. .