Abstract
As a vital negative regulator of Wnt signaling pathway,human Notum(hNotum)plays a crucial regula-tory 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 imag-ing 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 pmol/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 ab-normal 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.
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