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

The interaction between -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 A 2 ), and inhibiting such interac- tions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently con- tain "hot spots," small patches that are the main mediators of binding affinity. By making tight inter- actions with a hot spot, a small molecule can com- pete with a protein. The Tcf3/Tcf4-binding surface on-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 -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 collec- tions, when no biochemical functional assay is avail- able, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibi- tors of protein-protein interactions. Proteins 2006;