Disrupting Apicoplast DNA Replication in Plasmodium: Identifying Key Proteases for Antimalarial Therapy

Malaria remains a global health crisis, causing over 600,000 deaths annually, with most fatalities occurring in children under five. Plasmodium falciparum, the causative agent of malaria, relies on the apicoplast for key biosynthetic functions like isoprenoid production. The apicoplast is indispensable for parasite survival, making it a prime target for antimalarial therapies. Prex, a polyprotein containing DNA primase, helicase, and polymerase domains, is crucial for replicating the apicoplast genome. However, the proteases responsible for processing Prex into its functional subunits remain unidentified, representing a critical gap in our understanding of apicoplast DNA replication. Preliminary studies have identified three candidate proteases that potentially interact with Prex. This proposal will now focus on characterizing these proteases to better understand their role in apicoplast function and biogenesis. Aim 1 will confirm the localization of these proteases within the apicoplast, assess their importance for parasite growth and Prex maturation, and determine their complete set of interacting proteins. Aim 2 will characterize the biochemical properties of the proteases, using targeted and unbiased approaches to identify their substrates and map cleavage sites. This research will provide essential insights into proteolytic processes within the apicoplast, regardless of whether the candidate proteases directly process Prex. The findings will inform future high-throughput screens for small-molecule inhibitors targeting these proteases and support the identification of new therapeutic targets for novel antimalarial therapies.