Inhibition of protein–protein interactions: The discovery of druglike β‐catenin inhibitors by combining virtual and biophysical screening

The interaction between β‐catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 Å2), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain “hot spots,” small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4‐binding surface on β‐catenin contains a well‐defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to β‐catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4‐competitive compounds with the tightest binder having a KD of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein–protein interactions. Proteins 2006. © 2006 Wiley‐Liss, Inc.