Defining Nuclear H2O2 Regulation by Covalent Regulators

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Project Summary Loss of metabolic homeostasis is through to contribute to aging and replicative senescence. Control of redox balance is critical for maintenance of metabolic homeostasis. Improper levels of reactive oxygen species (ROS) are thought to be an important contributor to multiple aging related diseases such as metabolic diseases, cancer and neurodegenerative disorders. At high levels, ROS modify nucleic acids–an important mechanism by which this group of reactive metabolites leads to genomic instability and replicative senescence. While there has been much investment into understanding how different ROS damage nucleic acids, surprisingly little is known about the pathways which generate, detoxify and sense nuclear ROS. This knowledge-gap hampers our understanding of the roles nuclear ROS plays during biological aging. A major obstacle to deciphering the biology of nuclear ROS is the inability to control the levels of ROS in a nucleus-specific manner, through defined mechanisms of action. Small molecules have been instrumental in biological breakthroughs often regulating biological processes at a level of specificity and precision not achievable with even the most advanced genetic models. The purpose of this application is to develop a suite of chemical probes that specifically increase nuclear ROS levels and characterize their corresponding protein targets. We will do so, by combining a nuclear-localized H2O2 sensor (HyPer7) with a chemical proteomic-guide small molecule screen. We have previously used these approaches to identify a small inhibitor that increases C elegans longevity by ~45% and characterized its target protein, providing support for the great utility of chemoproteomic screening approaches to study biological aging. Here, we will leverage a cysteine-focused small molecule library (5000+ chemically diverse chloroacetamide/acrylamides) to identify covalent probes that increase steady state nuclear H2O2 levels. We focus on cysteines given their critical role in protein function and the ability to identify covalent inhibitors that engage them using chemoproteomics. A preliminary screen of 270+ molecules has already furnished 9 compounds that specifically increase nuclear H2O2 levels but not at other compartments. We will subsequently use chemical proteomics to identify the corresponding protein target and determine their importance in regulating nuclear H2O2 levels and replicative senescence. The research proposed herein, takes full advantage of a series of recently developed methods: genetically encoded ROS reporters and chemical proteomics, which have previously been used in isolation, to be integrated into an effective approach to identify the pathways that control nuclear H2O2 levels. These studies will provide both a deeper understanding of the key pathways involved in nuclear ROS regulation and develop a much-needed suite of pharmacological agents to study how ROS in different compartments shapes biological aging.