It is widely accepted that the aberrant expression of developmental transcription factors in adult tissues can result in the misexpression of target genes and reactivation of developmental programs, thereby contributing to tumorigenesis and/or tumor progression. I propose that the disruption of the transcriptional activity of developmental transcriptional factors overexpressed in cancer can be an attractive mechanism for potential anti-cancer treatment. The approach through which I hope to disrupt transcription is through the inhibition of the protein-protein interactions between target transcriptional factors and their cofactors. To that end, I have chosen two separate transcriptional complexes each known to be misexpressed and play a causal role in cancer phenotypes: SIX1 and CtBP1. SIX1, a homeobox gene normally expressed during embryogenesis, requires the EYA family of co-activator proteins to activate transcription. CtBP1 is a co-repressor that binds to a conserved peptide motif found in multiple transcription factors (as well as the adenovirus E1A protein) to elicit its effect. Since single amino acid mutations have been shown to significantly disrupt each protein-protein interaction, I hypothesize that only a few residues play an important role in these interactions and that these residue interactions can be disrupted by small molecules leading to decreased protein-protein interactions. To identify small molecule inhibitors that target these complexes, I have developed an AlphaScreen assay to monitor the interaction between SIX1 and EYA2 or CtBP1 and E1A. In collaboration with the National Institute of Health Chemical Genomics Center (NCGC) using a high throughput screen, I have identified compounds that specifically inhibit either the CtBP-E1A or SIX1-EYA2 interactions. These compounds have been validated using secondary, specificity, and cell culture experiments. I plan to further determine the mechanism of action of these compounds through additional biophysical and structural techniques, such as crystallography and isothermal calorimetry. Concurrently, the NCGC is synthesizing a large number of analogues around the lead compounds to improve their potency and solubility, which may facilitate future efforts to characterize their mechanism of action and optimize the compounds. In addition, I am also developing a peptide-based inhibitor of CtBP and its transcription factor partners to inhibit CtBP-mediated tumorigenic and metastatic properties.