title: Yeast-based high-throughput screen identifies Plasmodium falciparum equilibrative nucleoside transporter 1 inhibitors that kill malaria parasites.
abstract: Equilibrative transporters are potential drug targets; however, most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64 560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2-2 μM). These nine compounds completely blocked [(3)H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5-50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5-50 μM). Wild-type (WT) parasite IC50 values were up to 4-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that, in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.
authors.forename: I J
authors.initials: IJ
authors.lastname: Frame
authors.forename: Roman
authors.initials: R
authors.lastname: Deniskin
authors.forename: Alison
authors.initials: A
authors.lastname: Rinderspacher
authors.forename: Francine
authors.initials: F
authors.lastname: Katz
authors.forename: Shi-Xian
authors.initials: SX
authors.lastname: Deng
authors.forename: Robyn D
authors.initials: RD
authors.lastname: Moir
authors.forename: Sophie H
authors.initials: SH
authors.lastname: Adjalley
authors.forename: Olivia
authors.initials: O
authors.lastname: Coburn-Flynn
authors.forename: David A
authors.initials: DA
authors.lastname: Fidock
authors.forename: Ian M
authors.initials: IM
authors.lastname: Willis
authors.forename: Donald W
authors.initials: DW
authors.lastname: Landry
authors.forename: Myles H
authors.initials: MH
authors.lastname: Akabas
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000962:Antimalarials / Q000737:chemistry / Q000494:pharmacology*; D061245:Axenic Culture; D001692:Biological Transport / Q000187:drug effects; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test; D057166:High-Throughput Screening Assays; D033701:Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D033703:Nucleoside Transport Proteins / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D053058:Trophozoites / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D000962:Antimalarials; C430711:ENT1 protein, Plasmodium falciparum; C413444:FUI1 protein, S cerevisiae; D033701:Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins; D033703:Nucleoside Transport Proteins; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; C001943:5-fluorouridine; D000241:Adenosine; D014529:Uridine