Quantitative high-throughput screening identifies small-molecule modulators of Wnt/β-catenin signaling

The Wnt/β-catenin signaling pathway plays an important role in development and tissue homeostasis, and its dysregulation is implicated in various pathologies, including cancer, fibrosis, and neurodegeneration. However, the discovery of small-molecule modulators of this pathway remains challenging due to the pathway’s inherent complexity, characterized by ligand redundancy, overlapping receptor usage, and compensatory downstream signaling. In this study, we optimized a cell-based LEF/TCF-β-lactamase reporter assay for quantitative high-throughput screening in a 1536-well format. Screening 1280 compounds from the Library of Pharmacologically Active Compounds alongside 88 compounds from the Tox21 collection identified twelve potential antagonists of Wnt/β-catenin signaling. Follow-up studies confirmed the activity of 10 compounds, demonstrating consistent activity across two independent reporter systems (β-lactamase and luciferase). Western blot analysis showed that all compounds except for cytosine-1-beta-D-arabinofuranoside and PMEG reduced accumulation of both non-phosphorylated β-catenin (active) and total β-catenin, providing orthogonal validation of pathway inhibition. The identification of known Wnt inhibitors such as emetine, tyrphostin A9, niclosamide, ouabain, and podophyllotoxin further validated the assay’s robustness. Collectively, this study establishes a robust 1536-well screening platform for identifying Wnt pathway modulators and identifies topotecan, amsacrine, brefeldin A, and tyrphostin AG 879 as candidate small-molecule antagonist, thereby expanding the chemical tools for investigating Wnt/β-catenin signaling.