The First NIR Fluorescent Probe for Bioimaging of Cystathionine-γ-lyase Activity in Living Cells and Xenograft Tumor Models

Cystathionine γ-lyase (CSE) is a pivotal enzyme in the transsulfuration pathway and the biosynthesis of hydrogen sulfide, with strong implications for regulating cellular redox homeostasis and tumor progression. Real-time and <i>in situ</i> detection of CSE activity in living systems remains a significant challenge. To address this, we herein report a novel near-infrared (NIR) fluorescent probe, NIR-CSE, for the selective and sensitive monitoring of CSE in living cells and xenograft tumor models. The probe features an NIR fluorophore Cy-NH₂ tethered to l-homocysteine (recognition unit) methyl ester via a self-immolative carbamate linker. Upon sequential ester hydrolysis and specific cleavage by CSE, an intramolecular cyclization-elimination reaction is initiated, resulting in a strong NIR fluorescence enhancement (25-fold) with excitation/emission maxima at 690/720 nm. NIR-CSE demonstrated high selectivity over related enzymes and excellent sensitivity with a detection limit of 0.928 U/L. In living-cell imaging experiments, the probe successfully differentiated breast cancer cells from normal mammary epithelial cells based on endogenous CSE activity, with fluorescence signals predominantly localized in mitochondria. Furthermore, NIR-CSE visualized the posttranslationally enhanced enzymatic activity of CSE under diverse oxidative stress stimuli, such as H₂O₂, lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). Notably, NIR-CSE enabled, for the first time, <i>in vivo</i> fluorescence imaging of intratumoral CSE activity in breast cancer xenograft mouse models with a high signal-to-noise ratio (approximately 10). These findings established NIR-CSE as a powerful chemical tool for investigating CSE biology in living cells and animal models.