In vitro activity: iCRT3 is a potent small molecule inhibitor of the Wnt pathway that binds to β-catenin interferes with its interaction with TCF. iCRT3 significantly reduces the LPS-induced Wnt/β-catenin activity and also inhibits TNF-α production and IκB degradation in a dose-dependent manner. iCRT3 does not influence the transcriptional activity of FOP-Flash luciferase reporter, which harbors mutations in the DNA binding sites for TCF (β-cat response element), showing specificity of the response. It inhibits cytokine production in LPS-stimulated macrophages.

Kinase Assay: iCRT3 is an inhibitor of Wnt/β-catenin signaling with an IC50 of 8.2 nM in the Wnt responsive STF16-luc reporter assays.

Cell Assay: cells (HCT116) were cultured in McCoy's 5A medium supplemented with 10% FBS. Cells were treated with specified concentrations of compounds for 1 d and lysed in 50 μL cell lysis buffer at 75℃ for 10 min.