Dissecting multidrug resistance pathways in Trypanosomatids

项目摘要 抗锥虫药物究竟如何杀死寄生虫，在很大程度上仍不清楚。锥体虫 感染，以非洲锥虫、美洲锥虫和利什曼原虫的形式，直接 导致超过10亿人的疾病和贫困。因此，知识的差距对人类有重大影响。 冲击公认的治疗方法是硝呋莫司、依氟鸟氨酸、苄硝唑、喷他脒、苏拉明和 美拉胂醇都具有复杂的治疗方案、宿主毒性和迅速增长的耐药性。高 美拉胂醇的宿主毒性（1/4的患者出现脑病）使得新药的开发势在必行， NECT（硝呋莫司-依氟鸟氨酸复方制剂，静脉注射）和非昔硝唑（口服）已对此作出回应 治疗尽管取得了这一进展，但非昔硝唑的细胞死亡和耐药机制尚不清楚 和其他重要药物。基于ORFeome的布氏锥虫功能获得文库是 用于鉴定锥虫的直接药物靶点和耐药机制的最新工具。 已发表的美拉胂醇功能获得筛选的发现确定了耐药性的新方面 （包括线粒体蛋白）。来自非昔硝唑基因筛查的未发表数据表明， 抗性幸存者来自一组可明确识别的基因的诱导表达，这仍然是未知的。 阐明。多种杀锥虫药物聚集在同一组细胞学表型上，表明它们具有共同的细胞学表型。 细胞死亡的途径。根据遗传筛查数据和基于细胞学的表型，该提案将测试 抗锥虫药物具有共同的细胞杀伤和利用机制， 这些途径可以促进对广泛使用的疗法的多重和泛耐药性。在AIM 1中，所有临床上 相关的抗锥虫药物（硝呋替莫司、依氟鸟氨酸、苄硝唑、喷他脒、苏拉明和 fexinidazole）将接受GoF基因筛选和验证，以确定一组促进多基因和 泛耐药多种药物汇聚在锥虫氧化还原和线粒体功能上。AIM 2将 使用基因编码的荧光生物传感器来测试抗锥虫药物 处理扰乱了细胞质和细胞质中的氧化还原代谢和ROS应激管理， 我们对药物诱导的氧化还原应激的理解存在差距。T.布氏杆菌与一系列 建立的表型，AIM 3将确定多抗性基因如何促进细胞死亡表型 包括：细胞周期、DNA损伤和线粒体功能丧失。该研究具有较高的 因为它将把杀锥虫表型与它们的相关基因和遗传途径联系起来， 第一次其表达促进多药耐药的基因将阐明导致多药耐药的途径。 这些寄生虫的细胞死亡这些研究的发现将阐明细胞死亡的机制， 所有现有的锥虫疗法，并确定改进药物设计的目标。该提案将使 在未来几年内，在抗寄生虫化合物的开发和筛选方面做出更明智的选择。

PROJECT SUMMARY Precisely how anti-trypanosomatid drugs kill parasites remains largely unknown. Trypanosomatid infections, in the form of African trypanosomes, American trypanosomes, and Leishmania spp., directly contribute to disease and poverty of over 1 billion people. Thus, gaps in knowledge have a significant human impact. The well-established treatments nifurtimox, eflornithine, benznidazole, pentamidine, suramin, and melarsoprol all suffer from complex treatment regimens, host toxicity, and burgeoning drug resistance. The high host toxicity of melarsoprol (encephalopathy in ¼ of patents) made development of new drugs an imperative, which has been answered by NECT (nifurtimox-eflornithine combination, intravenous) and fexinidazole (oral) therapies. Despite this progress, mechanisms of cell death and drug resistance are unknown for fexinidazole and other significant drugs. The ORFeome-based Trypanosoma brucei Gain-of-Function Library is the state-of- the-art-tool for identification of both direct drug targets and mechanisms of drug resistance in trypanosomatids. Discoveries from a published melarsoprol Gain-of-Function screen identified novel aspects of resistance (including mitochondrial proteins). Unpublished data from a fexinidazole genetic screen demonstrated that drug resistant survivors arise from induced expression of a clearly identifiable set of genes, which remain to be elucidated. Multiple trypanocidal drugs converge on the same set of cytology phenotypes, suggesting shared pathways to cell death. Based on genetic screening data and cytology-based phenotypes, this proposal will test the central hypothesis that anti-trypanosomatid drugs share common mechanisms of cell killing and utilize pathways that can promote multi- and pan-resistance against widely used therapies. In AIM 1, all clinically relevant anti-trypanosomatid drugs (nifurtimox, eflornithine, benznidazole, pentamidine, suramin, and fexinidazole) will undergo GoF genetic screening and validation to identify a set of genes that promote multi- and pan-drug resistance. Multiple drugs converge on trypanosomatid redox and mitochondrial functions. AIM 2 will use genetically encoded fluorescent biosensors to test the working hypothesis that anti-trypanosomatid drug treatments perturb redox metabolism and ROS stress management in the cytosol and mitochondrion, filling a gap in our understanding of drug-induced redox stress. Drug cytotoxicity in T. brucei is associated with a set of established phenotypes, AIM 3 will determine how multi-resistance genes contribute to cell death phenotypes including: cell cycle, DNA damage, and loss of mitochondrial functions. The proposed research is of high significance because it will link trypanocidal phenotypes with their associated genes and genetic pathways for the first time. Genes whose expression promotes multidrug resistance will elucidate the pathways that lead to cell death in these parasites. Discoveries arising from these studies will illuminate mechanisms of cell death for all existing trypanosomatid therapies and identify targets for improved drug design. This proposal will enable better informed choices in anti-parasitic compound development and screening for years to come.