Abstract Osteosarcoma (OS) is the most common type of primary bone cancer and the third most common cancer in children and adolescents. Current combination modalities have improved OS treatment, but did not have a major improvement on patient survival. Thus targeting underlying molecular events can provide dramatic benefits especially for the treatment of patients with fatal metastatic disease. Recently it is shown in a mouse model of OS that high ezrin expression is linked to pulmonary metastasis, providing an early survival advantage for cancer cells reaching the lung. Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) family is conserved through evolution both structurally and functionally. By regulating membrane-cytoskeleton complexes, it plays key roles in normal cellular processes like maintenance of membrane dynamics, survival, adhesion, motility, cytokinesis, phagocytosis and integration of membrane transport with signaling pathways. Due to its critical role in these cellular functions and relevance to metastasis, ezrin is a validated molecular target for prevention of metastasis in OS. In order to identify small molecules that directly interact with recombinant ezrin protein, we screened a small molecule library by surface plasmon resonance technology (Biacore). Initial hits from this primary screen were then evaluated in functional assays. We tested our small molecules for inhibiting ezrin mediated chemotaxis in cell culture, mimicking ezrin morpholino phenotype in zebrafish development, inhibiting ezrin phosphorylation and ezrin-actin interaction, effecting xenopus embryo development and inhibiting lung metastasis of osteosarcoma cells in a mouse lung organ culture assay. Results from these secondary functional assays, binding kinetics values from Biacore experiments and drugability of the chemical structures allowed us to pick two lead compounds. In summary, we discovered two lead compounds that directly bind to ezrin protein with ∼10uM affinity and inhibit its function in multiple independent assays. These molecules form the foundation of new potential therapeutic agents for osteosarcoma metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1550.