Reaction-based fluorogenic probes for detecting protein cysteine oxidation in living cells

While “turn-on” fluorescent probes for hydrogen peroxide are well-established, tools for visualizing the products of oxidant reactions directly on proteins have been lacking. In this work, Ferreira et al. introduce a new class of reaction-based fluorogenic probes (e.g. CysOx1, CysOx2) that selectively detect protein cysteine sulfenic acid (Cys-SOH) in live cells. The probes combine favorable attributes—good cell permeability, rapid kinetics, high selectivity, low toxicity—and generate fluorescence only upon reaction with sulfenic acid. The authors adapted this chemistry into a high-throughput cellular assay and screened a kinase-inhibitor library. They found that inhibition of kinases (including the GSK3 family) correlates with elevated cysteine sulfenation. Proteomic mapping of treated cells revealed that redox changes localize to regulatory cysteines on antioxidant enzymes, linking kinase activity to modulation of the “sulfenome.” This work provides powerful chemical tools and reveals how small molecules may regulate cysteine oxidation in cellular redox networks.