Mutational analysis of an antimalarial drug target, PfATP4

Swaksha Rachuri

doi:10.17918/00010437

Malaria, caused by the Plasmodium parasite species and transmitted by the bite of an infected female Anopheles mosquito, remains a significant global health concern. Countries in sub-Saharan Africa bear a disproportionate burden of malaria, contributing to thousands of deaths among pregnant women and children under the age of five. Despite considerable efforts to combat the disease, challenges such as drug resistance, vector control, and limited effective vaccines persist. Therefore, the identification of new antimalarial drug targets and the development of novel therapeutics is of paramount importance in the ongoing fight against malaria. Among new antimalarials discovered over the past decade are multiple chemical scaffolds that target Plasmodium falciparum P-type ATPase (PfATP4). This essential protein is a Na+ pump responsible for the maintenance of Na+ homeostasis. PfATP4 belongs to the type 2D subfamily of P-type ATPases, for which no structures have been determined. To gain better insight into the structure/function relationship of this validated drug target, we generated a homology model of PfATP4 based on SERCA, a P2A-type ATPase, and refined the model using molecular dynamics in its explicit membrane environment. This model predicted several residues in PfATP4 critical for its function, as well as those that impart resistance to various PfATP4 inhibitors. To validate our model, we developed a genetic system involving merodiploid states of PfATP4 in which the endogenous gene was conditionally expressed, and the second allele was mutated to assess its effect on the parasite. Our model predicted residues involved in Na+ coordination, protein assembly/folding as well as the phosphorylation cycle of PfATP4. Phenotypic characterization of these mutants involved assessment of parasite growth, localization of mutated PfATP4, response to treatment with known PfATP4 inhibitors, and evaluation of the downstream consequences of Na+ influx. Our results were consistent with the model's predictions of the essentiality of the critical residues. Additionally, our approach confirmed the phenotypic consequences of resistance-associated mutations as well as a potential structural basis for the fitness cost associated with some mutations. Taken together, our approach provides a means to explore the structure/function relationship of essential genes in haploid organisms.

Mutational analysis of an antimalarial drug target, PfATP4

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Mutational analysis of an antimalarial drug target, PfATP4

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