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Abstract：
Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.
摘要：
锥虫硫酮是锥虫中的主要硫醇氧化还原载体，其生物合成和利用途径包含独特的酶，这些酶包括针对该家族中人类寄生虫的合适药物靶标。限速酶的过表达，γ-谷氨酰半胱氨酸合成酶（GSH1），可以增加细胞内锥虫酮的浓度。Melarsoprol直接抑制锥替酮，并预测对下游氧化还原生物学的影响，包括需要进一步调查的ROS管理和dNTP合成。因此，我们假设美拉索洛尔治疗会抑制DNA合成，使用BrdU掺入测定和细胞周期分析进行了测试。此外,我们分析了依氟鸟氨酸的作用,与锥虫硫酮途径接口，非西硝唑,由于对DNA合成的预测影响，和戊脒作为实验对照。我们发现，melarosoprol处理导致细胞周期停滞和24小时内DNA合成的完全抑制，通过GSH1过表达得到缓解。相比之下,分析的其他药物对DNA合成有更微妙的影响,但GSH1表达并未显著改变.这些发现共同暗示DNA合成作为治疗靶标，值得在抗锥虫药物的开发中进一步研究。
