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

非昔硝唑存活基因暗示一种新的基于氧化还原的耐药机制

• 批准号: 10573978
• 负责人: Galadriel Astra Hovel-Miner
• 金额: $ 24.23万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10573978
• 关键词: African; African Trypanosomiasis; American Leishmaniasis; Antiparasitic Agents; Benznidazole; Cell Cycle; Chagas Disease; Clinical; Clinical Trials; Combined Modality Therapy; DNA; DNA Damage; DNA biosynthesis; Data; Drug Metabolic Detoxication; Drug Targeting; Drug resistance; Drug resistance pathway; Enzymes; Evaluation; Family; Free Radical Formation; Future; Genes; Genetic; Genetic Screening; Giardiasis; Glutathione S Transferase A; Grant; Heterocyclic Compounds; Human; In Vitro; Infection; Knowledge; Laboratories; Leishmania; Leishmaniasis; Libraries; Lipids; Medicine; Metabolism; Metronidazole; Mitochondria; Molecular; Mutation; Nifurtimox; Nitroreductases; Open Reading Frames; Oral; Outcome; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Peroxidases; Persons; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Prodrugs; Proteins; Publications; RNA Interference; Reactive Oxygen Species; Recommendation; Research; Resistance; Risk; Role; Specificity; Stress; Sulfhydryl Compounds; System; Testing; Therapeutic; Trichomonas Infections; Trypanocidal Agents; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Work; Xenobiotics; clinically significant; comparative; cytotoxic; dithiol; drug development; drug mechanism; fexinidazole; gain of function; global health; glutaredoxin; high reward; innovation; insight; knock-down; molecular drug target; mutant; new therapeutic target; novel; resistance gene; resistance mechanism; screening; stress management; success; tool

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.

项目摘要 非昔硝唑已成为非洲锥虫病的一线治疗药物， 单药治疗T.冈比亚布鲁氏菌感染。因为它目前在非洲锥虫中的成功 治疗方面，非西硝唑正在积极研究作为治疗美国锥虫病（恰加斯病）的方法 和利什曼病，目前全球感染人数超过1500万。非昔硝唑联合硝呋莫司（NECT 联合治疗，HAT）和苄硝唑（查加斯病）作为临床上重要的硝基芳香化合物 用于治疗锥虫感染。硝基杂环化合物作为前药， 硝基还原酶（NTR）酶促活化以产生被认为损害DNA，脂质， 和蛋白质。自然发生的和实验室耐药性和交叉耐药性已被记录， 在T。分别在布鲁氏菌NTR突变体和基于RNAi的敲低研究中。非昔硝唑是一种 这种化合物在临床试验中的成功使其能够快速获得批准，而很少有出版物对药物进行评价 杀锥虫表型、作用机制（MoA）或潜在的耐药性。分子量不足 对非昔硝唑的MoA和耐药模式的理解代表了一个重大的知识差距。到 填补这一空白，我们最先进的T将布氏杆菌功能获得文库应用于非昔硝唑的研究 初步研究确定了11个与生存相关的ORF，包括氧化还原酶， 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，所识别的基因支持了 新的非昔硝唑存活基因构成了一个基于氧化还原的途径，能够促进 硝基芳族药物耐药性与非昔硝唑的MoA的关系。这一探索性的建议将采取 在非昔硝唑治疗期间研究促进生存的基因的风险适中， 确定药物的分子靶点和耐药机制。具体目标是针对 回答两个重要的问题：非昔硝唑存活ORF是否对硝基芳香化合物特异？和做 Fexinidazole GoF存活基因构成了一个基于氧化还原的耐药途径？目标1将采用一套 创新T。布鲁氏菌功能获得性基因筛选，以确定已鉴定的非昔硝唑存活基因 （i）对非昔硝唑耐药具有特异性，（ii）对所有硝基芳烃具有普遍性，或（iii）广泛相关的ROS应激 管理AIM 2将测试工作假设，即非昔硝唑耐药性可能源于一种新的氧化还原- 基于耐药途径，通过药物耐药性和交叉耐药性的无偏表型分析， 定位、线粒体功能、细胞周期和DNA合成以及ROS和氧化还原。拟议 这项研究意义重大，因为它将确定与非昔硝唑的MoA和耐药性相关的基因， 机制，填补了这一一线治疗的关键知识空白。需要新的药物来对付所有 寄生锥虫感染和这项工作的发现，预计将导致替代 治疗方法和新的药物靶点。