Proteases are believed to play an important role in tumor cell invasion and metastasis through degrading the basement membrane and extracellular matrix. Using the degenerate oligonucleotide primers which were designed based on the conserved sequence homology, five independent serine protease cDNAs including a novel serine protease, membrane-type serine protease 1 (MT-SP1), were obtained from a cDNA library of a human prostatic cancer cell line, PC-3. We have developed an ecotin phage display system that presents fully functional ecotin on the surface of filamentous phage. In this study, using purified recombinant protease domain of MT-SP1 and this phage display system, we have identified potent inhibitors for MT-SP1. The three-dimensional structure of protease domain of MT-SP1 and these ecotin mutants can be superimposed with trypsin molecule in the trypsin-ecotin tetrameric complex using the resources of the Computer Graphics Laboratory. This structure-based approach would provide us useful information to design an improved macromolecular inhibitor of MT-SP1. A structure-based approach coupled to phage display would be employed to design MT-SP1 inhibitors through stepwise optimization.
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