Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage–pathway enzymes to form the required purine nucleotides. The P. falciparum genome encodes four putative ENTs (PfENT1–4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine-salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (< 10 µM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target.

Malaria is a critical public health issue in the tropical world, causing extensive morbidity and mortality. Infection by unicellular, obligate intracellular Plasmodium parasites causes malaria. The emergence of resistance to current antimalarial drugs necessitates the development of novel therapeutics. A potential novel drug target is the purine import transporter. Because Plasmodium parasites are purine auxotrophic, they must import purines from their host to fulfill metabolic requirements. They import purines via equilibrative nucleoside transporter 1 (ENT1) homologs. Recently, we used a yeast-based high-throughput screen to identify inhibitors of the P. falciparum ENT1 (PfENT1) that kill P. falciparum parasites in culture. P. berghei infection of mice is an animal model for human malaria. Because P. berghei ENT1 (PbENT1) shares only 60% amino acid sequence identity with PfENT1, we sought to characterize PbENT1 and its sensitivity to our PfENT1 inhibitors. We expressed PbENT1 in purine auxotrophic yeast and used radiolabeled substrate uptake to characterize its function. We showed that PbENT1 transports both purines and pyrimidines. It preferred nucleosides compared with nucleobases. Inosine (IC 50 5 3.7 mM) and guanosine (IC 50 5 21.3 mM) had the highest affinities. Our recently discovered PfENT1 inhibitors were equally effective against both PbENT1-and PfENT1-mediated purine uptake. The PfENT1 inhibitors are at least 10-fold more potent against PfENT1 than human hENT1. They kill P. berghei parasites in 24-hour ex vivo culture. Thus, the P. berghei murine malaria model may be useful to evaluate the efficacy of PfENT1 inhibitors in vivo and their therapeutic potential for treatment of malaria.

Edited by Mike Shipston 5 The abbreviations used are: ACT, artemisinin-based combination therapies; Ado, adenosine; ENT, equilibrative nucleoside transporter; MTS, methanethiosulfonate; MTSEA-biotin, methanethiosulfonate ethylammonium-biotin; MTSET ϩ , methanethiosulfonate ethyltrimethylammonium; MTSES Ϫ , methanethiosulfonate ethylsulfonate; PfENT1, P. falciparum equilibrative nucleoside transporter type 1; SCAM, substituted cysteine accessibility method; SDM, synthetic defined media; TM, transmembrane segment; XC 50 , concentration causing 50% maximal effect.Downloaded from each independent experiment were normalized to the lowest concentration of MTSEA-biotin. Each point is the average of independent experiments. Average and S.D. are shown, for some points error bars are smaller than the points and are not shown. Solid line is a four-parameter variable slope fit to the data by GraphPad Prism 7. The data for WT and the T353C mutant could not be fit unambiguously. XC 50 values, number of independent replicates, Hill slope, and uptake at 8 mM MTSEA-biotin relative to uptake with the lowest MTSEA-biotin concentration is reported in Table 4. Please note that the WT data in Fig. 2A is also shown in Figs. 4 and 7 to provide a point of comparison for the effects of MTSEA-biotin on the individual TM2 and TM10 Cys mutants.

No abstract