Dissecting multidrug resistance pathways in Trypanosomatids

项目总结 抗锥虫药物是如何杀死寄生虫的，目前在很大程度上尚不清楚。锥虫 以非洲锥体、美洲锥体和利什曼原虫的形式直接感染 造成10多亿人的疾病和贫困。因此，知识的差距对人类具有重大意义 冲击力。久负盛名的治疗药物有硝呋莫司、依氟鸟氨酸、苯硝唑、扑热息痛、苏拉明和 美拉索前列醇都受到复杂的治疗方案、宿主毒性和迅速增长的耐药性的影响。高潮 美拉索洛尔的宿主毒性(1/4的专利是脑病)使新药的开发势在必行。 已经用NECT(硝呋莫司-依氟鸟氨酸联合静脉注射)和非辛硝唑(口服)回答 治疗。尽管有这一进展，非辛硝唑的细胞死亡和耐药机制尚不清楚。 以及其他重要的药物。基于ORFeome的布氏锥虫增益功能文库 用于确定锥虫直接药物靶点和耐药机制的ART工具。 一项已发表的美拉索前列醇功能增益筛查的发现确定了耐药性的新方面 (包括线粒体蛋白)。来自费辛硝唑基因筛查的未发表数据表明，该药物 抗药性幸存者的产生源于一组明显可识别的基因的诱导表达，这些基因仍有待于 已澄清。多种杀锥虫药物集中在同一组细胞学表型上，这表明 细胞死亡的途径。基于遗传筛选数据和基于细胞学的表型，这项提议将测试 抗锥虫药物具有共同的细胞杀伤和利用机制的中心假说 可促进对广泛使用的疗法产生多重和泛耐药的途径。在AIM 1中，所有临床上 相关抗锥虫药物(硝呋莫司、依氟鸟氨酸、苯硝唑、扑热息痛、苏拉明和 费辛硝唑)将接受GOF基因筛选和验证，以确定一组促进多基因和多基因 泛耐药。多种药物汇聚在锥虫的氧化还原和线粒体功能上。目标2将 使用基因编码的荧光生物传感器来检验抗锥虫药物的工作假设 治疗扰乱了胞浆和线粒体的氧化还原代谢和ROS应激管理，填补了 我们对药物引起的氧化还原应激的理解存在差距。布氏毛滴虫的药物细胞毒性与一系列 已确定的表型，AIM 3将确定多耐药基因如何影响细胞死亡表型 包括：细胞周期、DNA损伤和线粒体功能丧失。提出的研究具有较高的研究价值。 具有重要意义，因为它将把杀锥虫表型与其相关基因和遗传途径联系起来 第一次。表达促进多药耐药性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。这项提议将使 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 Unpublished data from a fexinidazole genetic screen demonstrated that drug resistant survivors arise from induced expression of a clearly identifiable set of genes, which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.