title: Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1) for antimalarial drug development.
abstract: Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([(3)H]adenosine) and pyrimidines ([(3)H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 μM), compared to guanosine (14.9 μM) and adenosine (142 μM). For pyrimidines, thymidine had an IC50 of 183 μM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 μM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial drugs that target both falciparum and vivax ENT1 may be feasible.
authors: Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA.
authors: Roman
authors: R
authors: Deniskin
authors: Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA.
authors: I J
authors: IJ
authors: Frame
authors: Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA.
authors: Yvett
authors: Y
authors: Sosa
authors: Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA; Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA; Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA. Electronic address: myles.akabas@einstein.yu.edu.
authors: Myles H
authors: MH
authors: Akabas
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000962:Antimalarials / Q000494:pharmacology*; D004176:Dipyridamole / Q000494:pharmacology; D055808:Drug Discovery; D033721:Equilibrative Nucleoside Transporter 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D006151:Guanosine / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007288:Inosine / Q000494:pharmacology; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000469:parasitology; D016780:Malaria, Vivax / Q000188:drug therapy / Q000469:parasitology / Q000517:prevention & control; D033701:Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000378:metabolism; D010966:Plasmodium vivax / Q000187:drug effects* / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011687:Purines / Q000378:metabolism / Q000494:pharmacology; D011743:Pyrimidines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014529:Uridine / Q000494:pharmacology
chemical_list: D000962:Antimalarials; C430711:ENT1 protein, Plasmodium falciparum; D033721:Equilibrative Nucleoside Transporter 1; D033701:Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins; D015800:Protozoan Proteins; D011687:Purines; D011743:Pyrimidines; D006151:Guanosine; D007288:Inosine; D004176:Dipyridamole; D000241:Adenosine; D014529:Uridine
keywords: ACT, Artemisinin-based Combination Therapies; CQ, chloroquine; Drug development; EC50, concentration causing 50% of maximal effect; ENT, equilibrative nucleoside transporter; EV, empty vector; HTS, high throughput screen; IC50, concentration causing 50% inhibition; Malaria; NBMPR, 4-nitrobenzylthioinosine; Nucleoside/nucleobase transport; Parasite; PfENT1, P. falciparum ENT type 1; Plasmodium vivax; Purines; PvENT1, P. vivax ENT type 1; SDM, synthetic defined media; SNP, single nucleotide polymorphism; Single-nucleotide polymorphism (SNP); Transporter; WHO, World Health Organization; WT, wild type; hENT1, human ENT type 1