Characterization and structure-guided targeting of plasmodium falciparum apicoplast DNA polymerase - malaria remains a significant health challenge, potentially affecting 40% of the global…

The National Institute of Allergy and Infectious Diseases awarded William Marsh Rice University $409,024 on February 6, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to characterize the structure and mechanism of Plasmodium falciparum apicoplast DNA polymerase and identify druggable allosteric sites for development of new antimalarial inhibitors.

The research addresses drug-resistant malaria by targeting the apicoplast, a metabolically essential organelle unique to Plasmodium parasites. The apicoplast DNA polymerase (APPOL) is the sole polymerase that replicates the apicoplast genome and exhibits structural and functional properties distinct from other polymerases, including high-fidelity synthesis, efficient proofreading, and proficient strand-displacement activities. The project will profile APPOL structure and dynamics during various stages of catalysis to identify unique structural elements and novel allosteric sites suitable for selective and potent inhibitor development, building on preliminary data defining kinetic pathways of APPOL catalysis and high-resolution structures in apo and DNA-bound states.

The award runs through January 31, 2028, with performance at Rice University in Houston, Texas. Iowa State University of Science and Technology serves as a sub-grantee under this award, contributing to the research effort.

Characterization and structure-guided targeting of plasmodium falciparum apicoplast DNA polymerase - 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.