Activity-Based Ratiometric FRET Probe Reveals Oncogene-Driven Changes in Labile Copper Pools Induced by Altered Glutathione Metabolism

Abstract Copper is essential for life, and beyond its well-established ability to serve as a tightly-bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low molecular weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a tris[(2-pyridyl)methyl]amine (TPA) bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and GSH/GSSG ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader crosstalk between metal and redox pathways in health and disease with activity-based probes. Significance Copper is a required metal nutrient for life, yet its altered homeostasis is associated with many diseases. Thus, to develop new methods to help decipher copper biology, we present an activity-based ratiometric FRET probe that exploits a biomimetic, copper(I)-dependent cleavage reaction to enable imaging of loosely-bound, labile copper pools in cells with metal and oxidation state selectivity and a self-calibrating ratiometric response. Application of this technology to cellular models of cancer reveals that oncogene-driven changes in the metabolism of glutathione, a major cellular redox buffer, leads to a labile copper(I) deficiency. This work establishes the relevance of copper dysregulation to cancer metabolism and presages further opportunities for activity-based sensing in studies of metal biology.