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Medical College of Ohio, Urology Research Center, Department of Urology [J. C-W., R. S., E. S-J., A. W., S. H. S., J. J.], and Department of Physiology and Molecular Medicine, [J. C-W., S. H. S., J. J.], Toledo, Ohio 43614-5807

4 To whom requests for reprints should be addressed, at Urology Research Center, Medical College of Ohio, Toledo, OH 43614-5807. Phone: (419) 383-3691; Fax: (419) 383-3168; E-mail: jerzy{at}golemxiv.dh.mco.edu

Proteolytic activity driven by urokinase (uPA) is commonly recognized as an important factor in metastasis and angiogenesis. The eradication of unwanted uPA activity expressed by cancer cells results in the inhibition of metastasis and angiogenesis. Development of novel and highly selective uPA inhibitors could, therefore, produce new treatments of cancer. The ultimate goal of this work is the identification of novel and selective inhibitors of uPA suitable for this purpose. We have chosen plasminogen activator inhibitor(s) type 1 (PAI-1), which selectively inhibits the urokinase plasminogen activator (uPA). However, PAI-1 is not a stable molecule and converts itself into the latent form with a half-life in the range of t_1/2 = 1–2 h. This conversion is associated with a partial insertion of the reactive loop (P4–P10') into the PAI-1 molecule. In such a conformation, P1–P1' and other sites are not accessible for reaction with uPA. To conquer this hurdle, we have produced several PAI-1 mutants by replacing chosen amino acids with cysteine in the hope of creating disulfide bridges, which could make this insertion more difficult. On the basis of the known structure of active PAI-1, we have identified amino acids that can be substituted with a cysteine residue to produce disulfide bridges linking the top and bottom parts of strands A3 and A5 as well as sites within the helix-D region. We created a total of seven cysteine mutants via point mutation (two to six point mutations), generating possible sites for disulfide bridge formation at the top and bottom parts of A3 and A5, within the helix-D region, or by a combination thereof. Desired mutations were introduced by PCR using appropriate primers. The mutant forms of PAI-1 containing the chitin-binding intein tag were then purified using affinity chromatography wherein the intein tag is cleaved, leaving mutant PAI-1 protein. Cys mutations resulted in proteins with extended half-life of PAI-1 from 2 to >700 h depending on the mutant. Novel PAI-1 were fully functional against uPA and showed activity in the in vitro model of angiogenesis, e.g., in the inhibition of sprout formation. Such prolonged serpin activity, which is therapeutically desired in cancer treatment and Cys-mutated PAI-1, could launch a new class of novel anticancer agents.

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