Malaria parasites of the genus Plasmodium are responsible for 300-500 million cases of human malaria and cause about one million deaths every year. Resistance to all traditional antimalarial drugs, and early signs of weakening effectiveness of artimesinin-based drugs in SE Asia, further emphasizes a need for basic and translational research to identify new interventions to treat and to avoid malaria. The P. falciparum and the P. vivax parasite are single cellular pathogens with multiple compartmentalized genomes: The nuclear genome, the mitochondrial genome, and the apicolplast genome. The last is a chloroplast-like relict plastid that is essential for parasite survival. The survival and development of the parasite, in all its stages, is dependent on continual management of smooth unwinding, replication, and ligations of DNA molecules. These functions rely on topoismerase enzymes, which are known to be important targets of antimicrobial chemotherapy. The development of malaria topoisomerase inhibitors has been hampered by an inability to express the proteins in functional form. The Rathod laboratory has argued and shown that toxicity of malaria proteins in functional form can limit the ability of traditional heterologous expression systems, such as E. coli, to support expression of functional protein. We have used a cell-free wheat germ translation system to successfully express a number of malaria proteins in functional for, including the first P. falciparum topoisomerase II. In this project application, we will express all P. falciparum and P. vivax candidate topoisomerase and DNA gyrase gene products, test their catalytic activity, purify them to homogeneity, and develop miniaturized efficient assays for High Throughput Screening. These studies will set the stage for direct screening of chemical libraries to ultimately yield small molecules to prevent and to treat malaria.

Public Health Relevance

Malaria parasites infect over 500 million people and are responsible for over 1 million deaths per year, often due to drug resistant parasites. The search for new antimalarials will be greatly aided by a High Throughput Assay (HTA) for Plasmodium topoisomerases, since such enzymes are excellent targets for other infectious agents.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
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Mcgugan, Glen C
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University of Washington
Schools of Arts and Sciences
United States
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White, John; Dhingra, Satish K; Deng, Xiaoyi et al. (2018) Identification and Mechanistic Understanding of Dihydroorotate Dehydrogenase Point Mutations in Plasmodium falciparum that Confer in Vitro Resistance to the Clinical Candidate DSM265. ACS Infect Dis :
White, John; Rathod, Pradipsinh K (2018) Indispensable malaria genes. Science 360:490-491
Mudeppa, Devaraja G; Kumar, Shiva; Kokkonda, Sreekanth et al. (2015) Topoisomerase II from Human Malaria Parasites: EXPRESSION, PURIFICATION, AND SELECTIVE INHIBITION. J Biol Chem 290:20313-24
Kumar, Shiva; Krishnamoorthy, Kalyanaraman; Mudeppa, Devaraja G et al. (2015) Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein-protein interactions. Acta Crystallogr F Struct Biol Commun 71:600-8
Mudeppa, Devaraja G; Rathod, Pradipsinh K (2013) Expression of functional Plasmodium falciparum enzymes using a wheat germ cell-free system. Eukaryot Cell 12:1653-63
Mailu, Boniface M; Ramasamay, Gowthaman; Mudeppa, Devaraja G et al. (2013) A nondiscriminating glutamyl-tRNA synthetase in the plasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway. J Biol Chem 288:32539-52