Antimalarial Drug Development: Quantifying Efficacy of PfENT1 Inhibitory Drugs on TM4 Yeast Mutants

Science Center, Bent Corridor

Infection of humans with Plasmodia parasites through a mosquito vector causes malaria. The growing resistance to artemisinin derivatives validates the urgency for new antimalarial drug development. P. falciparum is the most deadly Plasmodium species. Plasmodium parasites are purine auxotrophs. They import purines from the host cells to generate the purine nucleotides necessary for cellular metabolic processes. Equilibrative nucleoside transporters (ENT) are a family of membrane transporters that transports purine nucleobases and nucleosides. The Plasmodium falciparum genome contains four ENT homologues, PfENT1-4. PfENT1 is the primary purine-import pathway for the parasite purine salvage pathway1. PfENT1 inhibition leads to parasite death presumably due to purine starvation. A novel yeast-based high-throughput assay was used to identify nine PfENT1 inhibitors . We sought to determine whether residues in the fourth transmembrane segment of PfENT1, TM4, lined the substrate translocation pathway and/or affected inhibitor binding. Fourteen consecutive amino acids, I125 to A138, were mutated, one at a time, to cysteine (Cys) and individually expressed in yeast. We measured each mutant’s affinity for three purines: hypoxanthine, inosine, and adenosine. The IC50 values (half maximal inhibitory concentration) of each mutant were in the μM range, similar to those of wild type (WT). Thus, the mutations were tolerated and did not impact PfENT1 function. We also measured the ability of the nine PfENT1 inhibitors to block [3H]adenosine uptake. The IC50 values for one or more of the nine compounds were significantly increased for several of the mutants, as determined by a one-way ANOVA (p<0.05). Q135C caused a statistically significant increase for several of the compounds. The IC50 values of A131C and A134C also differed significantly from WT. Otherwise the effects were rather sporadic except for G132C. None of the nine compounds blocked purine transport by the G132C mutant. This suggests that the mutation interferes with drug binding. However, because G132C showed functional purine transport, G132C alters drug binding but not for purine binding. Of note, V126C grew very slowly and did not import [3H]adenosine. Further studies will aim to characterize other domains of PfENT1 using similar techniques. Once a full understanding of how PfENT1 interacts with each of the drugs is achieved, one of the nine compounds may be selected for further pharmaceutical development.