dissertationThe dysregulation of proteinâ€"protein interaction (PPI) networks has been implicated in many diseases. Designing therapeutic small-molecule inhibitors of these interactions is a challenging field for medicinal chemistry. This work advances the techniques for discovering more potent PPI inhibitors through integration of computational and biochemical techniques. High-throughput screening using fluorescence polarization and AlphaScreen assays identified an acyl hydrazone-containing inhibitor of the β-catenin/Tcf4 PPI, a key mediator of the canonical Wnt signaling pathway. By removing the undesirable acyl hydrazone moiety, a new compound, 4-(5H-[1,2,5]oxadiazolo[3',4':5,6]pyrazino[2,3-b]indol-5-yl)butanoic acid, was developed to selectively inhibit the β-catenin/Tcf4 interaction. The ethyl ester of this compound was tested in zebrafish embryos and shown to inhibit Wnt signaling in vivo at 2 and 10 μM concentrations. Differences between the PPI interface and the active site of traditional targets add to the difficulty of discovering PPI inhibitors. Herein, the relationship between inhibitor potency and ligand burialâ€"defined as the fraction of the solvent accessible surface areas of the bound over unbound ligand, θlâ€"in the PPI surface was evaluated. A positive correlation between θl and inhibitor potency was discovered. However, this correlation was secondary to the strong nonbonding interactions. A study of five PPI targets with corresponding inhibitor-bound crystal structures also revealed that empirical scoring functions were slightly better at identifying known inhibitors out of the putatively inactive test set, and the Lamarckian genetic algorithm was more successful at pose prediction. Due to the nature of the PPI surface, directly targeting the binding site may be difficult. A novel combination of computational methods explored the druggability, selectivity, and potential allosteric regulation of PPIs. Solvent mapping confirmed that Tcf4, E-cadherin, APC and axin use the same binding site on β-catenin in different ways. Evolutionary trace analysis indicated that the region surrounding W504 of β-catenin might be a potentially allosteric site. Site-directed mutagenesis testing results for a W504I β-catenin mutant resulted in three-fold increased binding of Tcf4 to β-catenin over the wild-type. This new site is promising for the discovery of future allosteric inhibitors of the β-catenin/Tcf4 PPI. The combined results from these studies reveals ways to better design PPI inhibitors
