Abstract Ezrin is a member of the ERM (Ezrin, Radixin, Moesin) family of proteins that functions as a cross-linker between the plasma membrane and actin cytoskeleton, thus allowing the cell to regulate cytoskeletal dynamics in response to external stimuli. Ezrin plays important roles in cell motility, membrane trafficking, morphogenesis, adhesion, survival and apoptosis. Ezrin also participates in crucial signal transduction pathways. Overexpression of ezrin contributes to the aggressive cancer behavior in various tumor types especially by enhancing the metastatic phenotype. Thus, ezrin represents a promising molecular target for cancer therapy. In our earlier studies, we have established that NSC305787 can directly bind to ezrin and inhibit its activity, which results in reduced metastasis. Since NSC305787 shares significant structural similarities with some anti-malarial drugs, we tested 400 anti-malarial compounds including 200 drug-like compounds and 200 probe-like compounds for their potential to directly bind to ezrin protein. We used surface plasmon resonance technology (Biacore) to screen direct binding. In this initial screen, 12 compounds demonstrated binding to ezrin over background values and selected as primary hits. Detailed SPR analysis of primary hits resulted in 7 compounds with comparable KD values to that obtained with our lead compound NSC305787. Primary hits were also evaluated for inhibition of the cell motility of murine osteosarcoma (OS) cells K7M2 and K12 that express high and low levels of ezrin, respectively. Cell motility and chemotaxis assays were conducted in parallel with viability assays to rule out general toxicity. Selected anti-malarial compounds were effective in inhibiting the motility of OS cells with high ezrin compared to cells with low ezrin. In conclusion, several anti-malarial drugs may have new indications in cancer therapy by inhibiting ezrin. Citation Format: Haydar Celik, Abraham Kallarakal, Daisy Colon-Lopez, Jeffrey Toretsky, Jurgen Bosch, Aykut Uren. Evaluation of anti–malarial compounds as inhibitors of ezrin in osteosarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2014-3987