Yeast-Based High-Throughput Screen Identifies Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites
| Autor: | Ian M. Willis, Roman Deniskin, Olivia Coburn-Flynn, Myles H. Akabas, Robyn D. Moir, Alison Rinderspacher, David A. Fidock, I. J. Frame, Francine Katz, Sophie H. Adjalley, Donald W. Landry, Shi Xian Deng |
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| Rok vydání: | 2015 |
| Předmět: |
Purine
Adenosine Saccharomyces cerevisiae Proteins Auxotrophy Plasmodium falciparum Protozoan Proteins Gene Expression Nucleoside Transport Proteins Saccharomyces cerevisiae Equilibrative nucleoside transporter 1 Biochemistry Article Small Molecule Libraries Antimalarials Structure-Activity Relationship chemistry.chemical_compound medicine Nucleotide Trophozoites Uridine Nucleobase Nucleoside Nucleotide and Nucleic Acid Transport Proteins chemistry.chemical_classification biology Axenic Culture Genetic Complementation Test Biological Transport General Medicine biology.organism_classification Yeast High-Throughput Screening Assays chemistry biology.protein Molecular Medicine Nucleoside Gene Deletion medicine.drug |
| Zdroj: | ACS Chemical Biology. 10:775-783 |
| ISSN: | 1554-8937 1554-8929 |
| DOI: | 10.1021/cb500981y |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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