Infection with unicellular eukaryotic Plasmodium species parasites causes malaria. P. falciparum causes the most virulent form of malaria. Currently, artemisinin combination therapy (ACT) is the treatment of choice for infected individuals. The rise of artemisinin resistant P. falciparum in Southeast Asia makes it imperative to develop new antimalarial drugs. Malaria parasites are purine auxotrophs. They transport purine precursors from the host erythrocyte into the parasite via the P. falciparum Equilibrative Nucleoside Transporter 1 (PfENT1). In the parasite, purine salvage pathway enzymes modify the purine precursors to form the nucleotides needed for RNA and DNA synthesis and other cellular processes. At purine concentrations found in human blood (<10 M), PfENT1 knockout parasites are not viable in culture. Thus, PfENT1 inhibitors may function as potent antimalarial drugs. The goal of this project is to explore the therapeutic hypothesis that inhibition of PfENT1 will kill malaria parasites and provide a novel target for antimalarial drug development. We have developed a simple, robust yeast cell growth assay and used it in a high throughput screen (HTS) to identify PfENT1 inhibitors. 5-fluorouridine (5-FUrd) kills wild type Saccharomyces cerevisiae. Mutant fui1? yeast that lack the endogenous plasma membrane purine/uridine nucleoside transporter are 100 times more resistant to 5-FUrd. PfENT1 transports 5-FUrd. In the presence of 125 M 5-FUrd, PfENT1-expressing fui1? yeast will only grow if a PfENT1 inhibitor is present to prevent 5-FUrd uptake. In 384 well plates, the Coefficient of Variation was <6.2%, Signal Window > 12, and the Z' score > 0.80, indicating a highly robust assay. We screened a 64,500 compound library and identified 171 hits. We tested nine of the top hits in a series of secondary assays. All nine inhibited [3H]adenosine uptake into both PfENT1-expressing yeast and into erythrocyte-free trophozoite stage P. falciparum with IC50 values in the 2 - 40 nM range. The nine compounds, five distinct chemical scaffolds, do not kill yeast but do kill P. falciparum parasites in culture with IC50 values in the 5 - 55 M range. The goals of this application are 1) to improve the potency and selectivity of the PfENT1 inhibitors through medicinal chemistry; 2) to define the mechanism of action of the inhibitors and their impact on parasite biology and growth at various life cycle stages; 3) to test the efficacy of the inhibitorsin a mouse malaria model; and 4) to identify the inhibitor binding site and the conformation of PfENT1 to which the inhibitors bind. Successful completion of this project will determine the utility of targeting PfENT1 for antimalarial drug development and may identify compounds suitable for further development.

Public Health Relevance

Over 200 million people develop malaria occur each year that results in over 600,000 deaths and an enormous economic burden due to lost ability to work during periods of severe illness. The development of resistance to current first line antimalarial drugs, such as the artemisinin combination chemotherapies, makes it imperative to identify new antimalarial targets and drugs. The goal of this project is to investigate the therapeutic potentia of inhibitors of the primary malaria purine transporter to serve as novel antimalarial drugs.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1-IDM-S (02))
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Mcgugan, Glen C
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Albert Einstein College of Medicine
Schools of Medicine
United States
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Arora, Avish; Deniskin, Roman; Sosa, Yvett et al. (2016) Substrate and Inhibitor Specificity of the Plasmodium berghei Equilibrative Nucleoside Transporter Type 1. Mol Pharmacol 89:678-85
Deniskin, Roman; Frame, I J; Sosa, Yvett et al. (2016) Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1) for antimalarial drug development. Int J Parasitol Drugs Drug Resist 6:1-11
Van Voorhis, Wesley C; Adams, John H; Adelfio, Roberto et al. (2016) Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond. PLoS Pathog 12:e1005763
Frame, I J; Deniskin, Roman; Arora, Avish et al. (2015) Purine import into malaria parasites as a target for antimalarial drug development. Ann N Y Acad Sci 1342:19-28
Frame, I J; Deniskin, Roman; Rinderspacher, Alison et al. (2015) Yeast-based high-throughput screen identifies Plasmodium falciparum equilibrative nucleoside transporter 1 inhibitors that kill malaria parasites. ACS Chem Biol 10:775-83