Yeast-Based High-Throughput Screen Identifies <i>Plasmodium falciparum</i> Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites

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 <i>Plasmodium falciparum</i> (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 <i>fui1</i>Δ 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 [³H]­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 (<i>pfent1</i>Δ) parasites. <i>pfent1</i>Δ 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

<i>N</i>‑Aryl-2-aminobenzimidazoles: Novel, Efficacious, Antimalarial Lead Compounds

From the phenotypic screening of the AstraZeneca corporate compound collection, <i>N</i>-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood stage of <i>Plasmodium falciparum</i> (<i>Pf</i>). Medicinal chemistry optimization of the potency against <i>Pf</i> and ADME properties resulted in the identification of <b>12</b> as a lead molecule. Compound <b>12</b> was efficacious in the <i>P. berghei</i> (<i>Pb</i>) model of malaria. This compound displayed an excellent pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended survival of animals for over 30 days following a dose of 50 mg/kg in the <i>Pb</i> malaria model. Compound <b>12</b> retains its potency against a panel of <i>Pf</i> isolates with known mechanisms of resistance. The fast killing observed in the <i>in vitro</i> parasite reduction ratio (PRR) assay coupled with the extended survival highlights the promise of this novel chemical class for the treatment of malaria