Dissecting multidrug resistance pathways in Trypanosomatids

项目概要 抗锥虫药物到底如何杀死寄生虫仍然知之甚少。锥虫科 直接感染非洲锥虫、美洲锥虫和利什曼原虫 造成超过 10 亿人的疾病和贫困。因此，知识差距对人类具有重大影响。 影响。成熟的治疗方法包括硝呋替莫、依氟鸟氨酸、苯并硝唑、喷他脒、苏拉明和 Melarsoprol 都面临复杂的治疗方案、宿主毒性和迅速增长的耐药性。高 Melarsoprol 的宿主毒性（1/4 的专利中存在脑病）使得新药的开发势在必行， NECT（硝呋替莫-依氟鸟氨酸组合，静脉注射）和非昔硝唑（口服）已经解决了这个问题 疗法。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库是最先进的 用于鉴定锥虫的直接药物靶点和耐药机制的最先进工具。 已发表的 melarsoprol 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）。非昔硝唑基因筛查的未发表数据表明，该药物 耐药幸存者是由一组清晰可辨的基因的诱导表达产生的，这些基因仍有待进一步研究。 阐明了。多种杀锥虫药物集中在同一组细胞学表型上，表明共享 细胞死亡的途径。根据基因筛查数据和基于细胞学的表型，该提案将测试 中心假设是抗锥虫药物具有共同的细胞杀伤和利用机制 可以促进对广泛使用的疗法的多重和泛耐药性的途径。在 AIM 1 中，所有临床 相关抗锥虫药物（硝呋替莫、依氟鸟氨酸、苯硝唑、喷他脒、苏拉明和 fexinidazole）将接受 GoF 基因筛查和验证，以确定一组促进多和 泛耐药性。多种药物集中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的工作假设 治疗扰乱细胞质和线粒体中的氧化还原代谢和ROS应激管理，填补 我们对药物引起的氧化还原应激的理解存在差距。布氏锥虫的药物细胞毒性与一组相关 确定表型后，AIM 3 将确定多重抗性基因如何导致细胞死亡表型 包括：细胞周期、DNA 损伤和线粒体功能丧失。所提出的研究具有较高的 意义重大，因为它将杀锥虫表型与其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性的基因将阐明导致多药耐药性的途径 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 在未来几年的抗寄生虫化合物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 Unpublished data from a fexinidazole genetic screen demonstrated that drug resistant survivors arise from induced expression of a clearly identifiable set of genes, which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.