Inhibition of upa/upar-interaction by cyclic upa-derived peptides

Focusing of the urokinase-type plasminogen activator (uPA) to the cell surface via interaction with its specific receptor (uPAR, CD87) is an important step in the invasion/metastasis process of cancer cells. The linear peptide uPA]9_3i and its more stable disulfide-bridged cyclic form (cyclo19'31uPAi9_3i) efficiently compete with uPA for binding to cell-surface associated uPAR. Using cyclo19'31uPAi9,3i as the lead structure, we synthesized a series of cyclic peptides in which certain ammo acids of uPAi were deleted and/or replaced by other amino acids. However, all of these cyclic peptides proved to be ineffective in disturbing the interaction of uPAR with uPA. We further synthesized the retro-inverso form of cycloI9-31uPAi9_3i, expected to display a much higher biological availability as cyclo'9-31uPA 19.31 due to its high resistance to proteolytic degradation. The retro-inverso form of cyclo19-31uPA[9.3i did not inhibit uPA/uPAR-interaction as determined by both flow cytofluorometry and solid-phase uPA-ligand binding assays. Finally, we systematically substituted each amino acid in cyclo'9>3luPA 19.31 by the corresponding D-amino acid to increase the rigidity of the molecule due to the induction of structural elements such as -tum motifs. Five of the 13 synthesized peptides revealed inhibitory capacities comparable to cyclo19'3IuPAi9-3i, one peptide had moderate uPAR-binding activity. The remaining 7 peptides did not interact with human uPAR. At present, peptides are synthesized in which multiple amino acids are substituted by D-amino acids to further increase the rigidity and proteolytic stability of the uPA-derived peptides. In addition, the effects of cyclo19-31uPAi9-3i and its D-amino acidscontaining derivatives on the invasion/metastasis process of tumor cells are analyzed applying in vitro and m vivo model systems.