ENGINEERING OF THE RECEPTOR-BINDING REGION OF UPA

UROKINASE-TYPE PLASMINOGEN ACTIVATOR (UPA) IS A THROMBOLYTICPROTEASE THAT HAS BEEN IMPLICATED IN CERTAIN NONTHROMBOLYTICPHYSIOLOGICAL PROCESSES, MOST NOTABLY THE INVASIVE GROWTH OR MIGRATION OF METASTATIC CELLS AND OF MACROPHAGES. THE ROLE OF UPA IN CELL MIGRATION APPEARS TO INVOLVE BINDING OF THIS PROTEASE TO SPECIFIC CELL SURFACE RECEPTORS. IT IS HOPED TO ENGINEER UPA-DERIVED MOLECULES THAT ARE PROTEOLYTICALLY INACTIVE AND BIND WITH ENHANCED AVIDITY TO THE UPA RECEPTOR. THESE MOLECULES MAY SERVE AS COMPETITIVE INHIBITORS OF NATIVE UPA RECEPTOR BINDING AND IN THIS WAY BEUSEFUL THERAPEUTIC AGENTS BY INHIBITING PHYSIOLOGICAL PROCESSES REQUIRING RECEPTOR-BOUND UPA. IN PHASE I, A UPA-RECEPTOR-BINDING ASSAY WILL BE DEVELOPED. THIS ASSAY WILL INVOLVE YEAST SECRETION OF UPA MOLECULES ONTO NITROCELLULOSE FILTERS AND PROBING OF THESE FILTERS WITH (125)I-LABELED, PURIFIED RECEPTOR MOLECULES. THE ASSAYWILL BE DESIGNED TO BE USED IN PHASE II FOR RAPID SCREENING OF YEAST COLONIES SECRETING UPA MOLECULES THAT HAVE ALTERED RECEPTOR BINDING. A LIBRARY OF VECTORS WITH MUTATIONS WITHIN THE CDNA ENCODING THE RECEPTOR-BINDING REGION OF UPA WILL BE CONSTRUCTED IN ESCHERICHIA COLI. PHASE II WILL INCLUDE SCREENING THIS LIBRARY, FOLLOWING STRATEGIES LEADING TO THE ENGINEERING OF UPA MOLECULES WITH ENHANCED RECEPTOR BINDING, AND TESTING THE ABILITY OF THESE MOLECULES TO COMPETITIVELY INHIBIT BINDING BY WILD-TYPE MOLECULES. THE ABILITY OF THESE MOLECULES TO INHIBIT INVASIVE CELL MIGRATION WILL ALSO BE TESTED.