Investigating the role of Plasmodium falciparum equilibrative nucleoside transporter 1 in malarial physiology and anti-malarial activity

Abstract

Malaria remains a devastating global health problem, accounting for nearly 1 million deaths annually. The emergence of parasite resistance to formerly effective antimalarial drugs has necessitated the identification of new targets for drug development. The malarial purine metabolic pathway represents one such possible target. The malarial parasite Plasmodium falciparum is a purine auxotroph, dependent upon salvage of purine nucleosides and nucleobases from the host erythrocyte to support parasite growth. Malarial parasites are sensitive to inhibitors of key enzymes in the purine salvage pathway. Both the immucillin class of purine nucleoside phosphorylase inhibitors and the tubercidin class of adenosine kinase inhibitors have been shown to block growth of P. falciparum cultures in vitro. However, little is known regarding the pathways these inhibitors utilize to gain access to the parasite cytosol.;P. falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is thought to be the major route of purine transport across the parasite plasma membrane. We examined the ability of PfENT1 to transport natural purines as well as inhibitors of purine metabolism in a heterologous Xenopus laevis oocyte expression system. We demonstrate that oocytes rapidly metabolize adenosine but not hypoxanthine, making hypoxanthine the preferred substrate for PfENTI transport studies in oocytes. Substrate metabolism effects may explain conflicting results in the literature regarding the adenosine transport Km of PfENTI as well as its ability to transport nucleobases.;While PfENT1 is capable of transporting members of the tubercidin class of drugs, Immucillin H did not seem to be transported into the oocytes. None of the immucillin drugs tested inhibited PfENT1 transport of [3H]-hypoxanthine or [3H]-adenosine. However, [3H]-Immucillin H appeared to bind to PfENT1 expressing oocytes. Structure-activity studies using unnatural purine nucleobases and nucleoside analogues were performed to identify structural determinants for substrate recognition and transport by PfENT1. These results provide new insights into the mechanism by which potential anti-malarial drugs are transported into the parasite cytoplasm.;Sequencing of the PfENT1 gene from the genomic DNA of 10 independent parasite strains identified four polymorphisms within the PfENT1 protein sequence, including F36S, V1291, L133F, and F394L. Analysis of the effects of these polymorphisms upon PfENT1 transport showed that the position 394 F→L mutation resulted in a 3-fold decrease in PfENT1 Km for adenosine and hypoxanthine, while the other polymorphisms did not significantly alter PfENT1's substrate affinity. To further characterize structure/function relationships within the region surrounding the position 394 polymorphism, cysteine mutagenesis was performed at PfENT1 positions 390 through 405. Cysteine substitutions for glycines at positions 392 and 396 completely eliminated PfENTI mediated transport of adenosine. Treatment of the PfENT1 I397C mutant with MTSEA-Biotin completely blocked adenosine transport, and modification could be protected by the presence of high concentrations of hypoxanthine. This residue in the middle of the transmembrane segment is inferred to be, at least transiently, on the water-accessible surface of the protein. Cysteine substitutions at F390 and L393 altered adenosine transport but application of MTSEA-Biotin had no effect on transport by these mutants or any of the other cysteine mutants. These findings show that positions 390, 392, 393, 394, 396, and 397 within PfENTI are all involved in the transport function of this protein.