Rational engineering of a large Stokes shift fluorogenic sensor for sensing Notum in living systems and inhibitor screening.

Notum is a serine hydrolase that negatively regulates Wnt/β-catenin signaling and is implicated in diseases including colorectal cancer (CRC). Here we report an enzyme-activatable fluorogenic sensor for dynamic monitoring and multidimensional imaging of human Notum (hNotum) in complex biological systems. Through iterative optimization of the hemicyanine reporter and the enzyme-recognition motif, we identified Oc-HNBI as an optimal hNotum substrate. Oc-HNBI shows high selectivity, strong binding affinity, rapid response, a large Stokes shift (142 nm), and a high quantum yield, providing exceptional spatial resolution and high imaging performance. The fluorogenic sensor was successfully applied for in situ imaging of hNotum activity in various biological settings, including 3D cell spheroids, cancer tissue sections, organ tissues, and in vivo mouse models. Additionally, Oc-HNBI was adapted to develop a high-throughput screening assay for discovering hNotum inhibitors. Using this assay, we identified Sennoside A, a natural compound, as a potent hNotum inhibitor with potential anti-CRC properties. Overall, this study presents a reliable fluorescent tool for sensing and imaging hNotum activity in living systems. This tool has the potential to advance the understanding of hNotum's role in human diseases and contribute to anti-CRC drug discovery.