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Clinical and veterinary trypanocidal benzoxaboroles target CPSF3 [Microbiology]
Proceedings of the National Academy of Sciences of the United States of America ( IF 11.1 ) Pub Date : 2018-09-18 , DOI: 10.1073/pnas.1807915115
Richard J Wall 1 , Eva Rico 1 , Iva Lukac 1 , Fabio Zuccotto 1 , Sara Elg 1 , Ian H Gilbert 1 , Yvonne Freund 2 , M R K Alley 2 , Mark C Field 1 , Susan Wyllie 1 , David Horn 3
Affiliation  

African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization’s target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei. Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.



中文翻译:

临床和兽用杀锥虫苯并恶硼烷靶向 CPSF3 [微生物学]

非洲锥虫引起致命和被忽视的热带疾病,在人类中被称为昏睡病,在动物中被称为纳加纳病。目前的疗法是有限的,但幸运的是,有前景的疗法正处于先进的临床和兽医开发阶段,分别包括 acoziborole(AN5568 或 SCYX-7158)和 AN11736。这些苯并恶硼烷可能是世界卫生组织到 2030 年实现疾病控制目标的关键。它们的作用方式以前是未知的。我们开发了一个高覆盖率的过表达库,并在这里使用它来探索布氏锥虫的药物作用模式. 最初,使用具有已知靶标的抑制剂来选择耐药性并测试大规模平行文库筛选和全基因组作图;这有效地确定了已知目标并验证了该方法。随后,使用过表达筛选方法来鉴定苯并恶硼烷、切割和聚腺苷酸化特异性因子 3 (CPSF3, Tb927.4.1340) 的靶标。我们使用独立的过表达菌株验证了 CPSF3 核酸内切酶作为靶标。击倒提供了 CPSF3 的基因验证作为必要条件,并且 GFP 标记证实了预期的核定位。分子对接和基于 CRISPR-Cas9 的编辑展示了 acoziborole 如何特异性阻断寄生虫的活性位点和 mRNA 加工,但不能阻断宿主 CPSF3。因此,我们的研究结果为 CPSF3 作为锥虫中重要的药物靶点提供了遗传和化学验证,并揭示了抑制 mRNA 成熟作为杀锥虫苯并恶硼烷的作用方式。了解基于苯并氧硼的疗法的作用机制可以帮助开发改进的疗法,以及预测和监测耐药性(如果或何时出现)。

更新日期:2018-09-19
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