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Abstract：
Chemotherapy continues to have a major impact on reducing the burden of disease caused by trypanosomatids. Unfortunately though, the mode-of-action (MoA) of antitrypanosomal drugs typically remains unclear or only partially characterised. This is the case for four of five current drugs used to treat Human African Trypanosomiasis (HAT); eflornithine is a specific inhibitor of ornithine decarboxylase. Here, we used a panel of T. brucei cellular assays to probe the MoA of the current HAT drugs. The assays included DNA-staining followed by microscopy and quantitative image analysis, or flow cytometry; terminal dUTP nick end labelling to monitor mitochondrial (kinetoplast) DNA replication; antibody-based detection of sites of nuclear DNA damage; and fluorescent dye-staining of mitochondria or lysosomes. We found that melarsoprol inhibited mitosis; nifurtimox reduced mitochondrial protein abundance; pentamidine triggered progressive loss of kinetoplast DNA and disruption of mitochondrial membrane potential; and suramin inhibited cytokinesis. Thus, current antitrypanosomal drugs perturb distinct and specific cellular compartments, structures or cell cycle phases. Further exploiting the findings, we show that putative mitogen-activated protein-kinases contribute to the melarsoprol-induced mitotic defect, reminiscent of the mitotic arrest associated signalling cascade triggered by arsenicals in mammalian cells, used to treat leukaemia. Thus, cytology-based profiling can rapidly yield novel insight into antitrypanosomal drug MoA.
摘要：
化疗继续对减轻锥虫引起的疾病负担产生重大影响。不幸的是,抗锥虫药物的作用模式（MoA）通常仍不清楚或仅部分表征。目前用于治疗人类非洲锥虫病（HAT）的五种药物中的四种就是这种情况；依氟鸟氨酸是鸟氨酸脱羧酶的特异性抑制剂。这里,我们使用一组布鲁氏菌细胞检测来探测当前HAT药物的MoA。分析包括DNA染色，然后进行显微镜和定量图像分析，或流式细胞术；末端dUTP缺口末端标记以监测线粒体（动体）DNA复制；基于抗体的核DNA损伤位点检测；以及线粒体或溶酶体的荧光染料染色。我们发现美拉洛尔抑制有丝分裂；硝呋替莫降低线粒体蛋白丰度；喷他脒引发了动体DNA的进行性丢失和线粒体膜电位的破坏；苏拉明抑制了细胞分裂。因此，目前的抗锥虫药物扰乱不同和特定的细胞区室，结构或细胞周期阶段。进一步利用这些发现，我们显示推定的丝裂原激活的蛋白激酶有助于美拉洛尔诱导的有丝分裂缺陷，让人想起哺乳动物细胞中由砷化物触发的有丝分裂阻滞相关的信号级联，用来治疗白血病.因此，基于细胞学的分析可以快速产生新的见解抗锥虫药物MoA。
