My research project has been directed at understanding the detailed mechanisms of protease/inhibitor interactions at the molecular level. The knowledge gained from this study will assist us to design molecules through protein engineering efforts. For the past year, I have been focusing on redesigning ecotin, a serine protease inhibitor found in E. coli. My goal is to convert wild type ecotin to a potent inhibitor of urokinase, a serine protease which has been implicated in mediating processes such tumor growth and invasion. Using phage display technology, I have isolated a ecotin mutant which has 3000-fold higher affinity towards urokinase. We also demonstrated that treating mice that carried human prostate cancer with this ecotin mutant blocked both tumor growth and metastasis. Our next goal is to design a short peptide version of ecotin based on the known structure of ecotin. In ecotin, two peptide loops closely interact with proteases. Displaying the crystal structure of the ecotin protease complex with MidasPlus on the workstations at the Computer Graphics Laboratory helped us to determine exactly how these two loops could be joined and form a cyclic peptide. We displayed this structurally constrained, cyclic peptide on phage and found that this peptide phage inhibited urokinase with significant potency. Again, we demonstrated that structural information coupled with the use of interactive molecular graphics is invaluable for protein engineering and molecular design.
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