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Abstract：
Malaria remains a major public health threat for billions of people worldwide. Infection with obligate intracellular, unicellular parasites from the genus Plasmodium causes malaria. Plasmodium falciparum causes the deadliest form of human malaria. Plasmodium parasites are purine auxotrophic. They rely on purine import from the host red blood cell cytoplasm via equilibrative nucleoside transporters to supply substrates to the purine salvage pathway. We previously developed a high throughput screening assay to identify inhibitors of the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Screening a small molecule library identified PfENT1 inhibitors that blocked proliferation of P. falciparum parasites in in vitro culture. The goal of the current work was to validate a high-resolution model of PfENT1 predicted by the AlphaFold protein structure prediction program. We superimposed the predicted PfENT1 structure on the human homologue structure, hENT1, and developed a structure-based sequence alignment. We mutated the residues in PfENT1 aligned with and flanking the residues in hENT1 that interact with the purine analog, nitrobenzylthioinosine (NBMPR). Mutation of the PfENT1 residues Q135, D287, and R291 that are predicted to form hydrogen bonds to purine nucleosides eliminated purine and pyrimidine transport function in various yeast-based growth and radiolabeled substrate uptake assays. Mutation of two flanking residues, W53 and S290, also resulted in inactive protein. Mutation of L50 that forms hydrophobic interactions with the purine nucleobase reduced transport function. Based on our results the AlphaFold predicted structure for PfENT1 may be useful in guiding medicinal chemistry efforts to improve the potency of our PfENT1 inhibitors.
摘要：
疟疾仍然是全球数十亿人的主要公共卫生威胁。专性细胞内感染，疟原虫属的单细胞寄生虫引起疟疾。恶性疟原虫是人类最致命的疟疾。疟原虫寄生虫是嘌呤营养缺陷型。它们依靠通过平衡核苷转运蛋白从宿主红细胞细胞质中输入嘌呤来为嘌呤补救途径提供底物。我们先前开发了高通量筛选测定以鉴定恶性疟原虫平衡核苷转运蛋白1型(PfENT1)的抑制剂。筛选小分子文库鉴定了在体外培养中阻断恶性疟原虫寄生虫增殖的PfENT1抑制剂。当前工作的目标是验证AlphaFold蛋白质结构预测程序预测的PfENT1的高分辨率模型。我们将预测的PfENT1结构叠加在人类同源结构上，hENT1，并开发了基于结构的序列比对。我们突变了PfENT1中与hENT1中与嘌呤类似物相互作用的残基对齐和侧翼的残基，硝基苄基硫代肌苷（NBMPR）。预测与嘌呤核苷形成氢键的PfENT1残基Q135，D287和R291的突变消除了各种基于酵母的生长和放射性标记的底物摄取测定中的嘌呤和嘧啶转运功能。两个侧翼残基的突变，W53和S290也导致无活性蛋白。与嘌呤核碱基形成疏水相互作用的L50突变降低了转运功能。基于我们的结果,PfENT1的AlphaFold预测结构可用于指导药物化学努力以提高我们的PfENT1抑制剂的效力。
