Biophysical Survey of Small-Molecule β-Catenin Inhibitors: A Cautionary Tale

Abstract

The canonical Wingless-related integration site signaling pathway plays a critical role in human physiology, and its dysregulation can lead to an array of diseases. β-Catenin is a multifunctional protein within this pathway and an attractive yet challenging therapeutic target, most notably in oncology. This has stimulated the search for potent small-molecule inhibitors binding directly to the β-catenin surface to inhibit its protein-protein interactions and downstream signaling. Here, we provide an account of the claimed (and some putative) small-molecule ligands of β-catenin from the literature. Through in silico analysis, we show that most of these molecules contain promiscuous chemical substructures notorious for interfering with screening assays. Finally, and in line with this analysis, we demonstrate using orthogonal biophysical techniques that none of the examined small molecules bind at the surface of β-catenin. While shedding doubts on their reported mode of action, this study also reaffirms β-catenin as a prominent target in drug discovery.

Figure 1

Schematic of the Wnt signaling pathway in both the inactive (left) and active (right) state.

Figure 2

Structures of representative Wnt signaling inhibitors acting upstream (1–3) and downstream (4) of the DC.

Figure 3

Structure of β-catenin with PPI interfaces and hotspots, and key PPI partners. (A) Schematic of β-catenin (aa 001–781) with the unstructured N-terminal domain (001–126), ARD (127–682), C-terminal domain (683–781), and phosphorylation sites (oncogenic—red or tumor suppressing—green). The 12 repeats are numbered. The five main interaction hotspots are highlighted in pink, green, white, yellow, and red.^, The color coding is consistent with that of Figure 1. (B) Shows key interactions between the TCF/LEF hotspot and the unstructured region of the TCF protein (yellow sticks). (C) Shows key interactions between the BCL9 hotspot and the BCL9 protein (purple cartoon/sticks). β-Catenin is shown with surface representation and key hotspot residues are shown in white; residues contributing to PPI in TCF4 and BCL9 are shown in black.

Figure 4

Structures of small molecules proposed to target Wnt/β-catenin signaling via direct engagement/binding of β-catenin. The year they were reported is indicated on the abovementioned timeline. Above arrow: discovered in cell free assays and below arrow: discovered by whole cell, functional assays.