Characterization and Structure-guided Targeting of Plasmodium falciparum Apicoplast DNA Polymerase

About This Grant

Malaria remains a significant health challenge, potentially affecting 40% of the global population and causing 200 million new cases annually. While existing antimalarial drugs such as chloroquine and artemisinin have been effective, increasing cases of drug resistance underscore the need for novel therapeutic strategies targeting essential and unique biological processes of the Plasmodium parasite. The apicoplast, a non-photosynthetic organelle present in nearly all Plasmodium species, plays critical metabolic roles throughout the parasite’s life cycle. Targeting the apicoplast presents a highly specific and effective strategy for malaria control and treatment. The apicoplast contains its own DNA genome that is replicated by a dedicated A-family DNA polymerase (apPol). As the sole polymerase localized to the apicoplast, apPol exhibits unique structural and functional adaptations, including domain and loop insertions and high-fidelity synthesis, efficient proofreading, and proficient strand-displacement activities. However, the structure and mechanism of apPol remain poorly understood, and no highly potent and specific inhibitors for apPol have been developed. We hypothesize that targeting unique allosteric sites of apPol during its catalytic cycle will provide a promising avenue for new antimalarial drug development. This study aims to comprehensively characterize structural and mechanistic properties of apPol to identify druggable allosteric sites for selective and potent apPol inhibitors. In Aim 1, we will profile the structure and dynamics of apPol during various stages of catalysis to identify unique structural element and novel allosteric sites for its various function. As shown in our preliminary data, we have defined kinetic pathways of apPol catalysis and determined high- resolution structures of apPol in apo and DNA-bound states. We will further investigate the structure and dynamics of apPol during strand displacement DNA synthesis and exonuclease proofreading to uncover unique structural elements and dynamics relevant for inhibitor development. In Aim 2, we will develop specific inhibitors targeting allosteric sites in apPol through computational and biochemical screening. Our preliminary studies have identified allosteric inhibitors targeting a dynamic domain of apPol with micromolar affinity. We will conduct extensive screening and optimization to enhance the specificity and potency of inhibitors through structure-activity analysis. These inhibitors will be validated using biochemical and parasite-killing assays. Our proposed research holds great potential for antimalarial drug development while providing broader insights into DNA polymerase evolution and the therapeutic targeting of A- family polymerases in various diseases.