Eponemycin is a Streptomyces metabolite that blocks angiogenesis. Despite the potential of this anti-angiogenic agent for inhibiting tumor cell metastasis, little is known about its mechanism of action. The goal of the proposed studies is to elucidate the mechanism by which eponemycins exerts its angiostatic activity, and it is anticipated that these studies will lead to an increased understanding of angiogenesis. As the first step toward achieving this goal, a biotinylated eponemycin analog will be synthesized as a probe for the eponemycin binding proteins (EBPs). This analog will be used to isolate EBPs, presumably through covalent modification of its protein targets. EBPs will be cloned and systems to produce these proteins for structure and function studies will be developed. In addition, antibodies against the EBPs will be raised for western blot analysis and subcellular localization experiments. These antibodies will be used as reagents to explore the role of eponemycin in the cell cycle regulation. Specifically, EBP levels at different stages of the cell cycle will be monitored and the effect of anti-EBP on cell cycle progression will be examined. In addition, the gene encoding EBP will be transiently expressed in endothelial cells to analyze the effect of the protein on cell cycle progression. Other studies of the EBPs include examination of their phosphorylation state and, if the protein(s) are phosphorylated, identification of phosphorylated sites. To further characterize the role of EBPs, a library of anti-angiogenic ligands will be generated based on the predicted eponemycin pharmacophore.
