Discovery of anthraquinones as potent Notum inhibitors for treating osteoporosis by integrating biochemical, phytochemical, computational, and experimental assays

Osteoporosis, a severe systemic skeletal disorder characterized by decreased bone mineral density, leads to increased risks of bone fragility and fracture. Although some herbal medicines (HMs) are clinically used for treating osteoporosis, the crucial anti-osteoporotic constituents and their mechanisms have not been well-elucidated. Notum, a negative regulator of Wnt/β-catenin signaling, has been validated as a druggable target for enhancing cortical bone thickness and alleviating osteoporosis. Herein, we showcase an efficient strategy for uncovering the key anti-Notum constituents from HMs via integrating biochemical, phytochemical, computational, and cellular assays. Following screening the anti-Notum potentials of HMs, <i>Polygonum multiflorum</i> Thunb. (PM), a commonly used anti-osteoporosis herb, showed potent and competitive inhibition against Notum. Phytochemical profiling coupling with docking-based virtual screening suggested that three anthraquinones, including rhein, emodin, and chrysophanol, showed high binding-potency towards Notum. Biochemical assays validated that three anthraquinones were strong competitive inhibitors of Notum, while rhein was the most potent one (IC₅₀ = 9.98 nM). Cellular investigations demonstrated that rhein markedly promoted osteoblast differentiation in dexamethasone-challenged MC3T3-E1 osteoblasts, while RNA sequencing showed that rhein remarkably regulated Wnt signaling-related and osteogenic differentiation-related genes. <i>In vivo</i> tests showed that rhein displayed favorable safety profiles in healthy mice and this agent significantly elevated bone mineral density, and augmented trabecula and cortical bone thickness in dexamethasone-induced osteoporotic mice. Collectively, this study showcases an efficient strategy for uncovering the key anti-Notum constituents from HMs, while rhein was identified as a naturally occurring Notum inhibitor that shows favorable safety profiles and impressive anti-osteoporosis effects.