Rational construction of a practical enzyme-activatable fluorogenic substrate for hNotum and its applications in functional imaging and inhibitor screening

Abstract

Notum, a negative regulator of the Wnt signaling cascade via removing the essential palmitoleate moieties on the Wnt proteins, has been validated as a promising target for treating various Wnt/β-catenin related diseases. Herein, a rational substrate design strategy was used to construct a highly specific and high-affinity fluorogenic substrate for real-time sensing and functional imaging of hNotum in complex biological systems. Utilizing computer-aided substrate design and phenotyping assays, 3-O-methyl-6-O-octanoylfluorescein (MOF) was selected as an ideal enzyme-activatable fluorogenic substrate for hNotum from a set of ester derivatives of the fluorophores bearing an optically adjustable phenolic group. Under physiological conditions, MOF could be readily hydrolyzed by hNotum to release a brightly fluorescent product 3-O-methylfluorescein (3-MF), which could be easily captured by the devices equipped with fluorescence detector(s). Further studies showed that MOF displayed good performance for sensing hNotum in complex biological samples with rapid response, ultrahigh sensitivity, excellent specificity, and high binding affinity (Km = 101.4 nM). MOF also exhibited excellent performance for in-situ functional imaging of hNotum in living cells, cancerous tissues, and tumor-xenograft mice. MOF was then used to construct a fluorescence-based biochemical assay for high-throughput screening (HTS) of hNotum inhibitors, while isoalantolactone (IAL) was identified as a potent hNotum inhibitor and an efficacious activator of Wnt signaling cascade from an in-house compound library. Collectively, this study devised the first-generation fluorogenic substrate for sensing and imaging hNotum activities in various biological systems, which offered a practical tool for deciphering the biological roles of hNotum and its relevance to human diseases.