Rationally engineered IR-783 octanoate as an enzyme-activatable fluorogenic tool for functional imaging of hNotum in living systems

As a vital negative regulator of Wnt signaling pathway, human Notum (hNotum) plays a crucial regulatory role in the progression of many human diseases. Deciphering the relevance of hNotum to human diseases requires practical and reliable tools for visualizing hNotum activity in living systems. Herein, an enzyme-activatable fluorogenic tool (IR-783 octanoate) was rationally engineered for sensing and imaging hNotum activity in living systems by integrating computer-aided molecular design and biochemical assays. IR-783 octanoate showed good optical properties, excellent specificity and high binding-affinity towards hNotum (Km = 0.98 µmol/L). IR-783 octanoate could be well up-taken into the cancerous cells or tumors that over-expressed organic anion transporting polypeptides (OATPs), and then hydrolyzed by cellular hNotum to release free IR-783 ketone, which created brightly fluorescent signals around 646 nm. Further investigations showed that IR-783 octanoate achieved a good performance for in-situ functional imaging of hNotum in both living cells, cancerous tissues and organs. It was also found that some SW620 cells with multipolar spindles could be stained by IR-783 octanoate to emit extremely bright signals, suggesting that this agent could be used as a novel visualizing tool for tracing the cells undergoing abnormal cell mitoses. Collectively, this study devises a highly specific fluorogenic tool for in-situ functional imaging of hNotum in living systems, which offers a practical and reliable tool to dynamically track the changes in hNotum activity under various conditions.