Tumor cell migration and metastasis in cancer are facilitated by interaction of the serine protease urokinase type plasminogen activator (uPA) with its receptor uPAR (CD 87). Overexpression of uPA and uPAR in cancer tissues is associated with a high incidence of disease recurrence and early death. In agreement with these findings, disruption of the protein-protein interaction between uPAR present on tumor cells and its ligand uPA evolved as an attractive intervention strategy to impair tumor growth and metastasis. For this, the uPAR antagonist cyclo[19,31][D-Cys(19)]-uPA(19)(-)(31) was optimized to efficiently interrupt binding of uPA to cellular uPAR. First, the disulfide bridge of this lead compound was shifted and then the modified peptide was shortened from the amino and carboxy terminus to generate cyclo[21,29][Cys(21,29)]-uPA(21)(-)(30). Next, cyclo[21,29][D-Cys(21)Cys(29)]-uPA(21)(-)(30) was yielded by changing the chirality of Cys(21) to D-Cys(21). For analysis of uPAR binding activity, we employed competitive flow cytofluorometric receptor binding assays, using FITC-uPA as the ligand and U937 promyeloid leukemia cells as the cellular source of uPAR. As demonstrated for cyclo[21,29][D-Cys(21)Cys(29)]-uPA(21)(-)(30), the achieved peptide modifications maintained receptor binding activity (IC(50) = 0.04 microM), which is close in order to that of the parent protein ligand, uPA (IC(50) = 0.01 microM). A detailed NMR analysis with restrained and free molecular dynamics calculations in explicit H(2)O exhibits a well-defined structure with characteristic features such as an omega-loop with two betaI-turns about Lys(3), Tyr(4), Ser(6), and Asn(7). Hydrophobic clustering of the side chains of Tyr(4), Phe(5), Ile(8), and Trp(10) is observed. Side chain mobility is analyzed with time-dependent distance restraints. The NMR structure of cyclo[21,29][D-Cys(21)Cys(29)]-uPA(21)(-)(30) is very similar to the previously reported structure of the amino terminal fragment of uPA. Systematic point mutations led to cyclo[21,29][D-Cys(21)Nle(23)Cys(29)]-uPA(21)(-)(30), which still binds to uPAR but is resistant to proteolytic cleavage, e.g., by the tumor-associated serine proteases uPA and plasmin, and is stable in blood serum or plasma. In conclusion, small cyclic peptides were created, which mimic the structure and activity of the binding epitope of uPA to uPAR and which may serve as novel therapeutic agents in cancer metastasis.