Yeast-Based High-Throughput Screen Identifies Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites
Yeast-Based High-Throughput Screen Identifies Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites
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DOI：
10.1021/cb500981y
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发表时间：
2015-03-01
影响因子：
4
通讯作者：
Akabas, Myles H.
中科院分区：
文献类型：
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作者：
Frame, I. J.;Deniskin, Roman;Akabas, Myles H.
平衡转运蛋白是潜在的药物靶标；然而，大多数功能测定涉及放射性底物吸收，不适合高通量筛选 (HTS)。我们开发了一种强大的基于酵母的生长测定法，可能适用于许多平衡转运蛋白。作为原理证明，我们将我们的方法应用于疟疾寄生虫恶性疟原虫 (PfENT1) 的平衡核苷转运蛋白 1。 PfENT1 抑制剂可能作为新型抗疟药物，因为 PfENT1 介导的嘌呤输入对于寄生虫增殖至关重要。为了鉴定 PfENT1 抑制剂，我们筛选了 64 560 种化合物，并通过它们在细胞毒性 PfENT1 底物 5-氟尿苷 (5-FUrd) 存在下挽救表达 PfENT1 的 fui1 Delta 酵母生长的能力鉴定了 171 种。在二次测定中，九种最高活性的化合物抑制以腺苷作为唯一嘌呤来源的嘌呤营养缺陷型酵母菌株的 PfENT1 依赖性生长（IC50 0.2-2 μM）。这九种化合物完全阻断表达 PfENT1 的酵母和无红细胞滋养体阶段寄生虫的 [H-3] 腺苷摄取 (IC50 5-50 nM)，并抑制氯喹敏感和耐药的寄生虫增殖 (IC50 5-50 muM)。与 PfENT1 敲除 (pfent1 Delta) 寄生虫相比，野生型 (WT) 寄生虫的 IC50 值低 4 倍。 pfent1 Delta 寄生虫杀灭表现出 WT 未观察到的延迟死亡表型。我们推断，在寄生虫中，这些化合物可以抑制 PfENT1 和具有相似功效的次要靶标。次要目标身份尚不清楚，但它的存在可能会降低寄生虫对 PfENT1 抑制剂产生耐药性的可能性。我们的数据支持这样的假设：通过 PfENT1 阻断嘌呤转运可能是抗疟药物开发的一种新颖且引人注目的方法。
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 fui1 Delta 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 mu M). These nine compounds completely blocked [H-3]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 mu M). Wild-type (WT) parasite IC50 values were up to 4-fold lower compared to PfENT1-knockout (pfent1 Delta) parasites. pfent1 Delta 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.