Respiratory failure due to pulmonary metastasis is the major cause of death for patients with osteosarcoma. However, the molecular basis for metastasis of osteosarcoma is poorly understood. Recently, ezrin, a member of the ERM family of proteins, has been associated with osteosarcoma metastasis to the lungs. The small molecule NSC 668394 was identified to bind to ezrin, inhibit in vitro and in vivo cell migration, invasion, and metastatic colony survival. Reported herein are the design and synthesis of analogues of NSC 668394, and subsequent functional ezrin inhibition studies. The binding affinity was characterized by surface plasmon resonance technique. Cell migration and invasion activity was determined by electrical cell impedance methodology. Optimization of a series of heterocyclic-dione analogues led to the discovery of compounds 21k and 21m as potential novel antimetastatic agents.
The most common cause for mortality in osteosarcoma (OS) is respiratory failure due to metastasis to lungs. Thus targeting underlying molecular events that lead to metastasis can provide a significant benefit to patients with fatal metastatic disease. Accumulating evidence from experimental animal models and human cases suggests that ezrin is a key factor in the metastasis of OS cells. Ezrin is a multifunctional protein that connects the actin cytoskeleton to extracellular matrix through transmembrane proteins. We screened small molecule libraries for compounds that directly interact with ezrin protein. Two small molecules that directly bind to ezrin with low micromolar affinity were selected based on inhibiting ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin-actin interaction, and ezrin mediated motility of OS cells in culture. These compounds were unique in their mechanism of action such that they inhibited in vitro phosphorylation of ezrin by protein kinase C (PKC) by binding to the substrate not by inhibiting the kinase activity of the enzyme. PKC was able to phosphorylate other substrates in the presence of these novel ezrin inhibitors. They also mimicked the ezrin morpholino phenotype and caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of osteosarcoma cells to mice, both molecules inhibited lung metastasis of ezrin-sensitive cells, but not ezrin-resistant cells. These two molecules demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis in osteosarcoma and other tumors with elevated ezrin in their metastatic subclones. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4505. doi:10.1158/1538-7445.AM2011-4505
Though advancements in chemotherapy and surgical techniques have ameliorated treatment of primary osteosarcoma (OS), the metastatic phenotype remains a clinical challenge. Overall five-year survival rates for patients with localized OS have improved to 60-70%, yet survival rates of patients with metastasis remains at 20-30% with mortality linked to metastatis-induced respiratory failure. Therefore, targeting fundamental molecular events that lead to metastasis may yield significant benefit to patients with OS. Accumulating evidence from clinical samples and pre-clinical animal models suggests that ezrin is a key regulator in the metastasis of OS cells. Ezrin is a multifunctional protein that connects the actin cytoskeleton to extracellular matrix through transmembrane proteins and a critical component for cell motility, adhesion and shape. We have recently identified that a small molecule, NSC 668394 acts as a potent and selective inhibitor of ezrin function and inhibits migration in both in-vitro and in-vivo models. Moreover, suppression of ezrin phosphorylation by NSC 668394 significantly reduced the metastatic behavior in cellular and animal models and has thus emerged as an important lead inhibitor. Consequently, we conducted a series of structure-activity-relationship (SAR) studies that monitored direct binding; OS cell migration and HUVEC monolayer invasion in ‘real-time’ with surface-plasmon resonance and electrical impedance technology. From our 2nd generation library, we have designed novel candidate inhibitors which feature enhanced ezrin-binding affinity, and improved anti-migration and anti-invasion activities. In addition to their potency against the different stages of OS metastasis, these compounds possess other desirable attributes for development including good druggable physical-chemical characteristics and are currently undergoing further preclinical evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3906. doi:1538-7445.AM2012-3906
Osteosarcoma is the predominant primary bone cancer in children with a low survival rate due to pulmonary metastasis. Research on the molecular mechanism driving pulmonary metastasis implicates the cytoskeletal protein ezrin as the critical component of the disease pathology. Ezrin links the actin cytoskeleton to the plasma membrane proteins, and its overexpression is associated with poor survival in patients and increased invasion in osteosarcoma cell lines. We used surface plasmon resonance technology to screen small molecule libraries for compounds directly binding to ezrin. Two molecules, NSC305787 and NSC668394, were chosen based on their ezrin binding affinities and ability to inhibit ezrin in multiple functional, biochemical, cellular, and in vivo assays. To test the anti-metastatic activity of these molecules in vivo, we utilized a tissue-specific transgenic mouse model expressing Cre recombinase to excise the Retinoblastoma (Rb) and p53 tumor suppressor genes in osteoblast progenitor cells. These animals develop spontaneous osteosarcoma with 100% penetrance and high frequency of liver and lung metastases, mimicking human disease. We tested the anti-metastatic potential of ezrin inhibitors that we discovered, NSC305787 and NSC668394, in this clinically relevant mouse model. We started the treatment on one group of animals at 2 months of age before any detectable tumor is present and on a second group of animals only after detection of smallest palpable tumor. Animals in each group were randomly assigned to one of three treatment options: DMSO (Control), NSC305787, or NSC668394. Animals were observed for primary tumor growth and metastasis formation. All animals were euthanized when primary tumor volume reached 2.0cm3, when animals had severe cachexia, or when animals showed signs of pulmonary insufficiency. Mice treated with NSC305787 and NSC668394 did not show any changes in the growth of their primary tumors and experienced similar survival times. However, our results suggest that NSC305787 inhibits osteosarcoma metastasis to lungs as confirmed by histopathological analysis. Therefore, when combined with adjuvant chemotherapy and surgical resection of the primary tumor, targeting ezrin with small molecules may yield a positive outcome and greater survival in a clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4874. doi:1538-7445.AM2012-4874
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.
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