Project Summary/Abstract Malaria control efforts are currently facing substantial challenges, primarily due to increasing drug resistance, including the first-line drugs artemisinins. From 2018 to 2021, there was a noticeable increase in new malaria cases, with the count rising from 237 million to 254 million, and the annual death toll surged from 567,000 to 617,000 in 2021. Over 90% of these cases and 96% of these deaths occurred in Sub-Saharan Africa, as reported by the World Health Organization (WHO). Tragically, 80% of the mortalities were children under the age of five. There is an urgent need for enhanced vector control measures and antimalarial drugs that are prophylactic, therapeutic, and transmission blocking. The proteasome plays a crucial role in regulating various cellular functions by degrading regulatory and damaged proteins. The proteasome is a well-established drug target for cancer treatment, with three FDA-approved drugs, and for certain infectious diseases, with two in clinical development. Malaria proteasome inhibitors have shown promise with killing activity against Plasmodium parasites at multiple stages of their life cycle, including the challenging transmission and liver stages. Therefore, a P. falciparum proteasome (Pf20S) inhibitor has potential to be a therapeutic, prophylactic, and transmission-blocking agent. Moreover, Pf20S inhibitors demonstrate synergistic effects when combined with artemisinins, making them even more appealing for malaria treatment. However, the development of orally bioavailable Pf20S inhibitors that can minimize the emergence of resistance while maintaining high selectivity for Pf20S over both the human constitutive proteasome and immunoproteasome presents a challenge. We have achieved significant progress in pursuit of these goals for antimalarial proteasome inhibitors. Furthermore, we have solved multiple cryo-EM structures of Pf20S with a β5 inhibitor, a β2 inhibitor, and a nonpeptide inhibitor, providing valuable insights into structure-activity relationships, mechanisms of resistance and collateral sensitivity. This application seeks to build upon these achievements by advancing our development program for both peptide-based and nonpeptide-based proteasome inhibitors, with a focus on enhancing potency, selectivity, and in vitro and in vivo pharmacokinetic properties to achieve oral bioavailability. We will conduct iterative structure-activity relationship (SAR) studies for both peptide- and nonpeptide-based Pf20S inhibitor series and assess their antimalarial activities at different parasite stages in vitro, as well as their parasitemia-reducing capabilities in animal models of malarial infection.