Emerging Artemisinin (ART) resistance in () poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug “Biperiden” that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive 3D7 and resistant 3D7k13 strains. Ring survival assays that are clinically relevant to analyze ART resistance in 3D7k13 parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites.

中文翻译：

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前折叠蛋白是抗青蒿素恶性疟原虫疟疾中未折叠蛋白反应的新型调节剂


()中出现的青蒿素 (ART) 耐药性对发现治疗抗 ART 寄生虫的新药提出了挑战。对 ART 抗性机制的集中研究表明，ART 抗性与涉及前折叠蛋白 (PFD) 的未折叠蛋白反应途径的表达上调存在密切的分子联系。然而，迄今为止，尚未确定 PFD 作为参与 ART 耐药机制的分子参与者的完整特征，也尚未发现阻止这一过程的小分子抑制剂。在这里，我们对所有 Prefoldin 亚基 (PFD1-6) 进行了功能性表征，并通过免疫沉淀耦合 MS/MS 分析证明了 PFD 在红细胞内寄生虫中的表达及其与 Kelch13 蛋白的相互作用，从而确定了 PFD 在 ART 耐药性中所起的致病作用。 PFD 所有亚基之间的系统生物物理相互作用分析揭示了它们形成复合物的潜力。 PFD 在 ART 抗性中的作用在直系同源酵母 PFD6 突变体中得到证实，其中酵母突变体中 PFD6 的表达使表型恢复为 ART 抗性。我们发现了 FDA 批准的药物“Biperiden”，它可以限制 Prefoldin 复合物的形成，并抑制其与其关键寄生虫蛋白底物 MSP-1 和 α-微管蛋白-I 的相互作用。此外，Biperiden 治疗可抑制 ART 敏感 3D7 和耐药 3D7k13 菌株中的寄生虫生长。与分析 3D7k13 寄生虫的 ART 抗性具有临床相关性的环存活测定证明了 BPD 抑制存活寄生虫生长的效力。总的来说，我们的研究提供了 PFD 在 ART 耐药机制中作用的第一个证据，并为耐药寄生虫的管理开辟了新途径。