3.1 ISX9 activates the Wnt/β-catenin signaling pathway
In order to identify novel small molecules that modulate the Wnt/β-catenin signaling pathway, a cell-based SuperTOPFlash reporter system was used to screen a known-compound library. Our screening identified ISX9, a small molecule with the isoxazole scaffold, as a potential activator of Wnt/β-catenin signaling (Figure 1A and B). To determine the agonistic effect on the Wnt/β-catenin pathway, the luciferase reporter SuperTOPFlash was transfected into HEK293T cells. Treatment with 2.5-40 µM ISX9 strongly increased the transcriptional activity of the SuperTOPFlash reporter in a dose-dependent manner. ISX9 at 40 µM could induce the reporter activity ~2700-fold above the basic levels (Figure 1C). However, ISX9 had little effect on the activity of negative control reporter SuperFOPFlash, which contains mutated TCF-binding elements. As anticipated, lithium chloride (LiCl), a well-known activator of Wnt signaling, remarkably increased the activity of the SuperTOPFlash reporter (Figure 1D).
To further confirm the effect of ISX9 on Wnt signaling, we performed the SuperTOPFlash reporter assay in human keratinocyte HaCaT cells. Similarly, ISX9 dose-dependently enhanced the activity of the SuperTOPFlash reporter in HaCat, while it did not affect the SuperFOPFlash reporter activity (Figure 1E). Together, these results demonstrated that ISX9 is capable of activating the Wnt/β-catenin pathway.