Development of Chemical Biology Tools to Modulate Redox Homeostasis and Study Subcellular Stress Responses

Intracellular redox balance is crucial for cell function and is primarily regulated by the relative concentrations of glutathione (GSH) and its oxidized dimer (GSSG). Multiple physiological processes, ranging from cell signaling to protein folding, depend on redox homeostasis. Therefore, several pathological conditions such as cancer, diabetes, and neurodegenerative diseases have been associated with redox imbalances. In eukaryotes, this homeostasis is controlled at the subcellular level, and each organelle possesses its own redox environment. Whereas the effects of oxidative stress have been thoroughly investigated, reductive stress has remained significantly underexplored. One way to understand this essential physiological process is to develop biocompatible probes to induce reductive stress and study the triggered responses. We envisioned that the GSH/GSSG ratio could be manipulated by direct reduction of the disulfide bond in GSSG to GSH using trialkylphosphine derivatives. We aim to achieve subcellular targeting by taking advantage of the local enzymatic activity of organelle-resident enzymes, triggering the release of tributylphosphine and a fluorescent reporter from a masked precursor. In this Thesis, we report the development of a mitochondria-targeted tributylphosphine probe to induce reductive stress in live cells and study the mitochondria-specific reductive stress response. A series of live-cell experiments, including fluorescence imaging and biological assays, showed that reductive stress is eventually transformed into oxidative stress through superoxide production. It activates the ATF4-ATF3-CHOP cascade, part of the integrated stress response, which upregulates the CHAC1 gene. CHAC1 plays a role in the regulation of glutathione and elevated expression of this gene promotes GSH depletion. Regulation of gene expression plays a crucial role in stress-response pathways. Commonly used reporter gene luminescent or colorimetric methods for use in live cells include assays based on secreted enzymes, luciferase, and GFP. These techniques often require exogenous substrates or lack sensitivity due to endogenous activity of isoenzymes or non-amplified GFP signal. We developed a HaloTag fluorescence reporter system for gene expression monitoring in live cells to complement these methods and address some of their limitations. The expression of the self-labeling HaloTag protein is correlated to that of the target gene through co-expression of both genes as non-altered separate proteins. Two fluorogenic HaloTag ligands with different spectral properties and binding kinetics label the HaloTag protein and result in a ratiometric fluorescence output. We envisioned that by adjusting the concentrations of the two fluorogenic ligands, the system could be tuned to reflect differential expression with a large dynamic range. As proof-of-principle, we chose to monitor the expression of the chaperone BiP upon induction of the unfolded protein response of the endoplasmic reticulum. Here, we describe the gene reporter design, the establishment of the reporter cell line using CRISPR-Cas9 gene-editing, the synthesis of fluorogenic HaloTag ligands, and preliminary live cells experiments for the reporter system validation. Building on the design of our original mitochondria-targeted probe, we also developed a series of tributylphosphine probes to modulate redox homeostasis in the endoplasmic reticulum. Here, we report the systematic approach to optimize the design of enzymatically activated ER-targeted tributylphosphines, their synthesis, and preliminary fluorescence imaging results. Altogether, this work demonstrated the importance of trialkylphosphines as chemical probes to modulate redox biology by developing strategies to tune their reactivity and target them to specific organelles. These probes potentially impact new therapies' development and enrich our understanding of the subcellular compartmentalization of redox signaling and organelle-specific stress responses