Abstract
Due to rising drug resistance, novel therapeutics are needed to combat the causative agent of malaria, Plasmodium spp.. Aspartic protease inhibition has emerged as a promising frontier for anti-malarial development as P. falciparum expresses five essential aspartic proteases in its symptomatic erythrocytic stages. These proteases play key roles in a diverse range of parasite processes including protein export, invasion, egress, and response to cellular stress. Previous studies have identified many inhibitors that kill Plasmodium in vitro through inhibition of one or more of these aspartic proteases. Yet few of these inhibitors have made it to clinical trials as anti-malarial therapeutics. We sought to identify novel compounds that target Plasmodium aspartic proteases. Herein, I utilize a conditional knockdown system to tune the endogenous expression of each of the five essential aspartic proteases in cultured P. falciparum, allowing us to perform in vitro target validation of both novel and disputed Plasmodium aspartic protease inhibitors. I used this technique to characterize the novel chalcone and predicted aspartic protease inhibitor AP8 as a primarily PM IX-targeting inhibitor, the first reported. Furthermore, I dive deeper into one of P. falciparum’s five essential aspartic proteases, signal peptide peptidase (SPP), a conserved ER-resident aspartic protease in eukaryotes that plays roles in secretion and the ER-associated degradation pathway. Several previous studies have identified inhibitors of the SPP-related enzyme γ-secretase with >100-fold selectivity to PfSPP vs. human SPP but have failed in animal models due to toxicity issues. Using a P. falciparum conditional knockdown of SPP, I confirm the mechanism of action of previously published inhibitors that inhibit PfSPP in trans-genera systems and kill Plasmodium in vitro. Using commercially available γ-secretase inhibitors with published in vivo safety profiling, I also identify additional compounds with anti-malarial activity in vitro that act through PfSPP inhibition. In my thesis work, I also investigated the effects of SPP inhibition or depletion on the parasite life cycle, proteome stress, and protein secretion. Taken together, this demonstrates the use of conditional knockdowns as a tool to perform moderate-throughput target validation. Furthermore, we identify and characterize novel lead compounds for anti-malarial development that should be the subject of further study.
Committee Chair
Daniel Goldberg
Committee Members
Davide Sibley; Heather True-Krob; Maggie Chen; Natalie Niemi; Usheer Kanjee
Degree
Doctor of Philosophy (PhD)
Author's Department
Biology & Biomedical Sciences (Molecular Cell Biology)
Document Type
Dissertation
Date of Award
8-13-2026
Language
English (en)
DOI
https://doi.org/10.7936/abr2-fv90
Recommended Citation
Floyd, Katherine Lillie, "Aspartic Protease Inhibition as an Anti-Malarial Strategy" (2026). Arts & Sciences Graduate Student Theses and Dissertations. 3835.
The definitive version is available at https://doi.org/10.7936/abr2-fv90