Project Summary/Abstract Copper is an essential metal nutrient that plays important roles in human health and disease. The potent redox activity of copper is required for a diverse array of physiological processes including respiration, antioxidant defense, and hormone and neurotransmitter biosynthesis and metabolism. However, dysregulated copper homeostasis can contribute to cancer, metabolic diseases like obesity and diabetes, and heart and neurodegenerative disorders, often through aberrant oxidative stress and damage pathways. We are developing new chemical reagents that enable copper-specific imaging in biological models to help decipher the contributions of copper to healthy and disease states, spanning the subcellular to cellular to animal level, along with chemoproteomics approaches to identify and target copper-dependent disease vulnerabilities. The scientific premise is that building a copper imaging and proteomics toolkit will provide foundational chemical reagents to both further our understanding of the roles of copper in physiology and pathology and open new avenues for therapeutic intervention. This competitive renewal application is driven by design innovations where we invent new generalizable, activity-based sensing platforms for copper detection and activity-based proteomics for copper-dependent disease targets, as well as scientific rigor in the synthesis and use of multiple characterization techniques to apply these tools in cell and animal models. Specifically, we will pursue protein - labeled copper probes to study how copper is stored, trafficked, and utilized at the subcellular and cellular level, chemiluminescent indicators to enable dynamic and longitudinal copper imaging at the tissue and animal level, and chemoproteomics approaches to identify copper-dependent druggable sites and covalent fragment lead compounds against these targets.