Fexinidazole survival genes implicate a novel redox-based mechanism in drug resistance

项目总结 非辛硝唑已经成为治疗非洲锥虫病的一线药物，提供了第一个口服 单一疗法治疗冈比亚布氏毛滴虫感染。因为它目前在非洲锥虫中取得了成功 治疗方面，正在积极研究非辛硝唑作为治疗美国锥虫病(Chagas)的方法。 以及利什曼病，目前全球有超过1500万人感染利什曼病。非辛硝唑联合硝呋莫司(NECT) 联合治疗，HAT)和苯硝唑(恰加斯病)作为临床上重要的硝基芳香族化合物 用于治疗锥虫感染。硝基杂环化合物的作用是需要 硝基还原酶(NTR)被酶激活以产生细胞毒性物质，这种细胞毒性物质被认为可以破坏DNA，脂质， 和蛋白质。自然产生的和实验室产生的耐药性和交叉耐药性已被记录在案， 分别在布氏锥虫NTR突变体和基于RNAi的击倒研究中。非辛硝唑是一种改用的 一种化合物，在临床试验中的成功使其迅速获得批准，但很少有出版物对其进行评估 杀锥虫性表型、作用机制(MOA)或抗药性潜力。分子不充分 对非辛硝唑的MOA和耐药模式的了解是一个重大的知识鸿沟。至 填补了这一空白，我们最先进的布氏锥虫功能增益库被应用于非辛硝唑的问题 耐药性和初步研究确定了11个与生存相关的ORF，包括氧化还原酶，a 过氧化物酶、二硫醇谷氧还蛋白和谷胱甘肽S转移酶。总的来说，识别出的基因支持 中心假设：新的非辛硝唑生存基因构成了一个基于氧化还原的途径，能够促进 硝基芳香族药物耐药性与非辛硝唑MOA的关系。这项探索性的建议将采取 在非辛硝唑治疗期间研究促进生存的基因的适度风险，以获得高回报 识别药物的分子靶点和耐药机制。具体目标是 回答两个重要的问题：非辛硝唑的存活ORF是不是硝基芳香族化合物特有的？并做到了 非辛硝唑Gof生存基因是否构成氧化还原耐药途径？AIM 1将采用一套 创新的布氏毛滴虫功能增益基因筛查以确定已识别的非辛硝唑生存基因 是(I)对非辛硝唑的抗药性，(Ii)对所有的硝基芳香族，或(Iii)广泛相关的ROS应激 管理层。Aim 2将验证以下工作假设：非辛硝唑耐药性可由一种新的氧化还原- 基于耐药途径通过无偏表型分析耐药和交叉耐药，蛋白质 定位，线粒体功能，细胞周期和DNA合成，以及ROS和氧化还原。建议数 这项研究意义重大，因为它将识别与非辛硝唑的MOA和耐药性相关的基因 机制，填补这一一线治疗的关键知识空白。需要针对所有人的新药 寄生锥虫感染和这项工作的发现预计将导致替代 治疗方法和新药靶点。

PROJECT SUMMARY Fexinidazole has emerged as the front-line treatment against African trypanosomiasis, offering the first oral monotherapy against T. brucei gambiense infections. Because of its current success in African trypanosome treatment, fexinidazole is actively being investigated as a therapy against American trypanosomiasis (Chagas) and Leishmaniasis, which currently infect over 15 million people globally. Fexinidazole joined nifurtimox (NECT combination therapy, HAT) and benznidazole (Chagas disease) as clinically significant nitroaromatic compounds for the treatment of trypanosomatid infections. Nitroheterocyclic compounds function as prodrugs that require nitroreductase (NTR) enzymatic activation to generate cytotoxic species that are thought to damage DNA, lipids, and proteins. Naturally occurring and laboratory resistance and cross-resistance have been documented based on T. brucei NTR mutants and in RNAi-based knock-down studies, respectively. Fexinidazole is a repurposed compound whose success in clinical trials enabled rapid approvals with few publications evaluating the drugs trypanocidal phenotypes, Mechanism of Action (MoA), or potential for drug resistance. Inadequate molecular understanding of fexinidazole’s MoA and modes of drug resistance represent a significant knowledge gap. To fill this gap, our state-of-the-art T. brucei Gain-of-Function library was applied to the question of fexinidazole resistance and preliminary studies identified 11 survival associated ORFs including oxidoreductases, a peroxidase, dithiol glutaredoxin, and a glutathione S-transferase. Collectively, the identified genes support the central hypothesis that novel fexinidazole survival genes constitute a redox-based pathway able to promote nitroaromatic drug resistance with implications for fexinidazole’s MoA. This exploratory proposal will take the modest risk of investigating genes that promote survival during fexinidazole treatment, toward the high reward of identifying the drugs molecular targets and drug resistance mechanisms. The Specific Aims are targeted to answer two significant questions: Are fexinidazole survival ORFs specific to nitroaromatic compounds? and Do fexinidazole GoF survival genes constitute a redox-based drug resistance pathway? Aim 1 will employ a set of innovative T. brucei Gain-of-Function genetic screens to determine if the identified fexinidazole survival genes are (i) specific to fexinidazole resistance, (ii) general to all nitroaromatics, or (iii) broadly associated ROS stress management. AIM 2 will test the working hypothesis that fexinidazole resistance can arise from a novel redox- based resistance pathway through unbiased phenotypic analysis of drug resistance and cross-resistance, protein localization, mitochondrial functionality, cell cycle and DNA synthesis, and ROS and redox. The proposed research is significant because it will identify genes associated with fexinidazole’s MoA and resistance mechanisms, filling critical knowledge gaps for this frontline therapeutic. New medicines are needed against all parasitic trypanosomatid infections and discoveries arising from this work are expected to result in alternative therapeutic approaches and new drug targets.