Most current small molecule drugs are enzyme inhibitors or ligand-like molecules. They compete with natural substrates or ligands in deep binding pockets. Signal transduction often involves protein-protein interactions. Drug development has achieved some success in disrupting protein-protein interactions such as p53-MDM2 binding using small molecules and peptide mimetics. Our recent work suggests that intramolecular interactions mediate auto-activating or auto-inhibitory effects on MDM2 and MDMX, thus have important roles in regulating p53 activity and stress response. Our findings reveal potential new targets for the therapeutic modulation of p53. Intramolecular interaction has also been implicated in the regulation of numerous other proteins. However, stabilizing protein intramolecular interaction, the opposite of disrupting protein-protein binding, is largely unexplored as a therapeutic approach. We propose to use MDMX as a model to investigate the feasibility of stabilizing protein intramolecular interaction for p53 activation. Two specific aims are proposed: (1) Identify peptides that stabilize MDMX intramolecular interaction by in vitro compartmentalization screen. (2) Develop a high throughput screen for small molecules that stabilize MDMX intramolecular interaction. This work will provide proof of concept and lead compounds for the development of new drugs that target an important tumor suppressor pathway. Lessons from this study will also be applicable to the therapeutic targeting of other important disease-associated proteins.

Project Narrative This proposal will develop methods to screen for short peptides and small molecules that promote the internal binding of two domains in the MDMX protein, thus stabilizing the MDMX conformation in an inactive state. Such compounds should lead to activation of the p53 tumor suppressor protein (a binding partner of MDMX), resulting in cell death or growth arrest of tumor cells. The project aims to generate lead compounds for further optimization and drug development. Furthermore, the methods developed for targeting MDMX can also be used to identify regulators of other disease-associated proteins, because intramolecular interaction occurs in many complex proteins and is important for their normal function.