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

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正在研究对常用的唑类抗真菌化合物的可能抗性机制。在农业和医药领域，真菌对唑类化合物的抗性发展是一个世界性的主要问题，造成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，作为农业和人类抗真菌治疗，唑类药物越来越失败：一种真菌的耐唑菌株的百分比在五年内从5%增加到20%，据报道，耐唑真菌感染的患者死亡率很高。这种抗性可能源于DNA的不稳定性，但对导致真菌DNA在唑治疗后发生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。这项提议的工作还将促进第一代经济条件较差的研究生的参与，加强与肯·马库斯教授（克莱姆森大学）的合作研究努力，并与威廉·彭宁顿教授（克莱姆森大学）合作打击化学中的隐性偏见。此外，Brumaghim教授将在为期一周的住宿夏令营中，通过教授DNA损伤实验室，向初高中学生介绍生物无机化学。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意义的基本生物过程背后的化学。这项工作的结果有可能促进对抗真菌耐药机制的理解，并指导未来的抗真菌开发。唑类化合物是作物保护中使用最广泛的一类杀菌剂，也是世界范围内治疗人类真菌感染的一线药物。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。唑耐药机制包括编码抗真菌靶蛋白的ERG11基因突变和编码唑外排泵的ERG11基因上调。唑抗性可能源于DNA不稳定和染色体拷贝数增加（非整倍体），但导致真菌DNA在唑处理后发生这些变化的机制尚不清楚。PI Brumaghim和co-PI Kozubowski的初步数据表明，尽管共享Erg11作为共同靶点，不同的唑类在人类病原体新型隐球菌中表现出广泛的耐药发展。此外，在体外，唑类药物氟康唑与铜和铁结合，增强活性氧（ROS）的产生，促进金属介导的DNA损伤。氟康唑也增加新生C.的ROS和细胞损伤。这项工作将验证一种假设，即由唑类抗真菌药物与铜和/或铁相互作用产生的ROS和DNA损伤是导致唑类耐药性的DNA损伤和遗传不稳定的一般机制。这一新的抗真菌机制将通过以下方法建立：1)定量测定化学上不同的唑类化合物在体外结合铁和铜、促进ROS生成和损伤DNA的能力；2)测定相同的唑类化合物在正常和高铜或铁条件下对酿酒酵母（Saccharomyces cerevisiae）和新生酵母（C. neoformans）两种模式真菌细胞ROS、DNA完整性和耐药性发展的影响。这项工作旨在确定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之间的相关性成为可能。通过研究azole-metal binding和DNA damage是azole抗真菌耐药性的基础，PI和co-PI提出了一个重要的实验问题，对真菌耐药性具有潜在的广泛的科学意义。该奖项反映了美国国家科学基金会的法定使命，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。

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.