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

Clemson University, Clemson SC

Investigators

Abstract

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.