Novel Coumarin-Based Fluorescent Probe for Tracking Cys, GSH, and Hcy Dynamics in Lysosomes under Oxidative and Hyperosmotic Stress

Lysosomal cysteine (Cys), glutathione (GSH), and homocysteine (Hcy) are vital biothiols essential for maintaining cellular redox homeostasis and regulating critical physiological functions. Aberrant levels of these biothiols are closely linked to various diseases. Understanding the dynamics of lysosomal biothiols is crucial for elucidating their physiological roles and involvement in disease progression. However, the simultaneous and selective detection of Cys, GSH, and Hcy in lysosomes remains challenging due to their structural similarities and dynamic behaviors. In this study, we developed Lyso-FP, a novel coumarin-based fluorescent probe tailored for lysosomal targeting and the simultaneous discrimination of Cys, GSH, and Hcy through three distinct emission channels. By integrating multiple binding sites with a lysosome-targeting moiety, Lyso-FP exhibits high specificity, sensitivity, and subcellular selectivity, enabling precise visualization of biothiol dynamics under oxidative and hyperosmotic stress conditions. Additionally, Lyso-FP was successfully employed for real-time monitoring of biothiol fluctuations in a zebrafish model of oxidative and hyperosmotic stress. This work introduces an innovative tool for studying lysosomal biothiol dynamics, offering critical insights into lysosome-associated processes and their roles in cellular physiology and pathology. Moreover, it presents a promising strategy for diagnosing and investigating lysosome-associated diseases, paving the way for future biomedical and therapeutic advancements.