Azole Antifungals Coordinate Metals and Create Reactive Oxygen Species That Damage DNA and Cause Chromosomal Instability

唑类抗真菌药协调金属并产生活性氧，从而损害 DNA 并导致染色体不稳定

• 批准号: 2203847
• 负责人: Julia Brumaghim
• 金额: $ 49.2万
• 依托单位: Clemson University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2203847&HistoricalAwards=false
• 关键词: Azole; Antifungals; Coordinate; Metals; Create

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Julia Brumaghim and Lukasz Kozubowski of Clemson University are studying possible resistance mechanisms to commonly used azole antifungal compounds. In both agriculture and medicine, fungal development of resistance to azole compounds is a major worldwide problem, causing crop loss and agricultural-to-human-pathogen fungal resistance. Due to resistance development, azoles are increasingly failing as both agricultural and human antifungal treatments: the percentage of azole-resistant strains of one fungus increased from 5% to 20% in five years, and high patient mortalities are reported for azole-resistant fungal infections. This resistance may stem from DNA instability, yet little is known about the mechanisms that lead to changes to fungi DNA upon azole treatment. Understanding the development of resistance to antifungal compounds will impact the broad areas of biology, chemistry, agriculture, and medicine. This proposed work also will promote participation of a first-generation, economically disadvantaged graduate student, strengthen collaborative research efforts with Professor Ken Marcus (Clemson), and combat implicit bias in chemistry in a collaborative effort with Professor William Pennington (Clemson). In addition, Professor Brumaghim will introduce middle school and high school students to bioinorganic chemistry through teaching a DNA damage lab as part of a week-long residential summer chemistry camp. This research will train next-generation interdisciplinary researchers as it explores the chemistry behind a fundamental biological process with global implications. Results of this work have the potential to advance understanding of antifungal resistance mechanisms and to guide future antifungal development.Azole compounds are the most widely used class of fungicides for crop protection as well as a first-line treatment for human fungal infections worldwide. Crop loss and agricultural-to-human-pathogen fungal resistance is a major global issue, since fungi are developing resistance to azole compounds. Azole resistance mechanisms include mutations in the ERG11 gene that encodes the antifungal target protein and upregulation of ERG11 or genes encoding azole efflux pumps. Azole resistance may stem from DNA instability and increases in chromosomal copy numbers (aneuploidy), yet the mechanisms that lead to these changes to fungi DNA upon azole treatment are unknown. Initial data from PI Brumaghim and co-PI Kozubowski indicate that despite sharing Erg11 as a common target, different azoles display a wide range of resistance development in the human pathogen Cryptococcus neoformans. In addition, the azole drug fluconazole binds to copper and iron, enhances reactive oxygen species (ROS) generation, and promotes metal-mediated DNA damage in vitro. Fluconazole also increases ROS and cellular damage in C. neoformans. The proposed work will test the hypothesis that ROS generation and DNA damage by azole antifungals interacting with copper and/or iron is a general mechanism for the DNA damage and genetic instability that causes azole resistance. This novel mechanism for azole antifungal resistance will be established by: 1) quantifying the ability of chemically diverse azole compounds to bind iron and copper, promote ROS generation, and damage DNA in vitro, and 2) determining the effects of the same azole compounds on cellular ROS, DNA integrity, and development of drug resistance in two model fungi Saccharomyces cerevisiae and C. neoformans under normal and elevated copper or iron conditions. This work aims to establish the role of metals and ROS in azole-mediated DNA damage and enable correlations of azole properties with their effects on DNA damage and resistance development. By investigating the hypothesis that azole-metal binding and DNA damage underlie azole antifungal resistance, the PI and co-PI is asking an important experimental question with potentially broad scientific implications for fungal resistance.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在化学系生命过程化学（CLP）项目的支持下，克莱姆森大学的Julia Brumaghim和Lukasz Kozubowski正在研究对常用唑类抗真菌化合物的可能耐药机制。在农业和医学中，真菌对唑类化合物的抗性发展是一个主要的世界性问题，导致作物损失和农业对人类病原体的真菌抗性。由于耐药性的发展，唑类作为农业和人类抗真菌治疗越来越失败：一种真菌的唑类耐药菌株的百分比在五年内从5%增加到20%，并且据报道唑类耐药真菌感染的患者死亡率很高。 这种耐药性可能源于DNA的不稳定性，但对唑类处理后导致真菌DNA变化的机制知之甚少。了解抗真菌化合物耐药性的发展将影响生物学，化学，农业和医学的广泛领域。这项拟议的工作也将促进第一代，经济上处于不利地位的研究生的参与，加强与教授肯马库斯（克莱姆森）的合作研究工作，并打击化学中的隐性偏见与教授威廉彭宁顿（克莱姆森）的合作努力。此外，教授Brumaghim将介绍初中和高中学生通过教学DNA损伤实验室的生物无机化学作为为期一周的住宅暑期化学夏令营的一部分。这项研究将培养下一代跨学科研究人员，因为它探索具有全球影响的基本生物过程背后的化学。唑类化合物是目前应用最广泛的一类杀菌剂，在作物保护和人类真菌感染的治疗中具有重要的应用价值。 作物损失和农业对人类病原体的真菌抗性是一个主要的全球性问题，因为真菌正在对唑类化合物产生抗性。唑类耐药机制包括编码抗真菌靶蛋白的ERG 11基因突变和ERG 11或编码唑类外排泵的基因上调。唑类耐药可能源于DNA不稳定性和染色体拷贝数增加（非整倍性），但唑类处理后导致真菌DNA发生这些变化的机制尚不清楚。来自PI Brumaghim和co-PI Kozubowski的初步数据表明，尽管Erg 11是共同的靶标，但不同的唑类药物在人类病原体新型隐球菌中显示出广泛的耐药性发展。此外，唑类药物氟康唑与铜和铁结合，增强活性氧（ROS）的产生，并促进金属介导的体外DNA损伤。氟康唑还增加C.新人类拟议的工作将测试这一假设，即与铜和/或铁相互作用的唑类抗真菌剂的ROS产生和DNA损伤是导致唑类耐药的DNA损伤和遗传不稳定性的一般机制。 这种唑类抗真菌药耐药性的新机制将通过以下方式建立：1）定量化学上不同的唑类化合物在体外结合铁和铜、促进ROS产生和损伤DNA的能力，和2）确定相同的唑类化合物对细胞ROS、DNA完整性和两种模式真菌酿酒酵母和C.正常和高铜或铁条件下的新型人。 这项工作的目的是建立金属和活性氧在唑介导的DNA损伤中的作用，并使唑的性质与它们对DNA损伤和抗性发展的影响相关。通过研究唑类抗真菌药耐药性的基础是唑类金属结合和DNA损伤的假设，PI和co-PI提出了一个重要的实验问题，该问题对真菌耐药性具有潜在的广泛科学意义。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。