Rational engineering of an optimized near-infrared fluorogenic sensor for efficient discovery of potent Notum inhibitors as anti-osteoporosis agents

Abstract

Notum, an important member of the serine hydrolase superfamily, acts as a key negative feedback regulator of the Wnt signaling. Inhibiting Notum activity has emerged as a feasible therapeutic strategy for treating osteoporosis (OP) via activating Wnt/β-catenin signaling. This work aimed to develop a high-performance near-infrared (NIR) fluorogenic sensor for high-throughput screening and characterization of anti-Notum agents. First, a dual-driven strategy combining computer-aided molecular design with enzymatic phenotyping assays was utilized to identify an NIR fluorogenic scaffold (HTCF) for engineering Notum substrates. A suite of HTCF esters were then synthesized, while HX-HTCF (the n-hexanoate ester of HTCF) was selected as an optimum substrate for Notum owing to its desirable NIR emission, rapid response, superior signal-to-noise ratio, ultrahigh sensitivity, and high affinity. HX-HTCF was subsequently used to construct a fluorescence-based biochemical assay for the highly efficient discovery of Notum inhibitors. H5 was identified as a novel potent Notum inhibitor, which strongly inhibited Notum-catalyzed HX-HTCF hydrolysis in a competitive manner. Further investigations showed that H5 significantly activated Wnt/β-catenin signaling and promoted osteoblast differentiation in dexamethasone-induced MC3T3-E1 osteoblasts. Collectively, this study devises a practical Notum-activatable NIR fluorogenic sensor, which immensely facilitates the discovery of Notum inhibitors as novel anti-osteoporosis agents.