The urokinase receptor (uPAR, CD87) as a target for tumor therapy: uPA-silica particles (SP-uPA) as a new tool for assessing synthetic peptides to interfere with uPA/uPA-receptor interaction.

Many different processes in the physiology and pathophysiology of human beings are regulated protein/protein interactions such as receptor/ligand interactions. A more detailed knowledge of the nature of receptor/ligand binding sites and mechanisms of interaction is necessary as well in order to understand the process of cancer spread and metastasis. For instance, the cell surface receptor uPAR (CD87) and its ligand, the serine protease urokinase-type plasminogen activator (uPA), facilitate tumor invasion and metastasis in solid malignant tumors. Besides its proteolytic function in activating the zymogen plasminogen into the serine protease plasmin, binding of uPA to tumor cell-associated uPAR initiates various cell responses such as tumor cell migration, adhesion, proliferation, and differentiation. Hence, the tumor-associated uPA/uPAR system is considered a potential target for cancer therapy. Here we briefly describe a new technology using micro-silica particles coated with uPA (yields SP-uPA) and reaction of SP-uPA with recombinant soluble uPAR (suPAR) to test the competitive antagonistic potential of synthetic uPA peptides by flow cytofluorometry (FACS). We discuss the data obtained with the SP-uPA system from two different points of view: (1) The enhanced potential of improved uPA-derived synthetic peptides compared to previously described peptides, and (2) comparison of the new technique to other test systems currently used to identify uPA/uPAR or other protein/protein interactions.

Synthesis, solution structure, and biological evaluation of urokinase type plasminogen activator (uPA)-derived receptor binding domain mimetics.

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.

uPA-silica-Particles (SP-uPA): a novel analytical system to investigate uPA-uPAR interaction and to test synthetic uPAR antagonists as potential cancer therapeutics.

The urokinase-type plasminogen activation system, including the serine protease uPA (urokinase-type plasminogen activator) and its cell surface receptor (uPAR, CD87), are important key molecules in tumor invasion and metastasis. Besides its proteolytic function, binding of uPA to uPAR on tumor cells exerts various cell responses such as migration, adhesion, proliferation, and differentiation. Hence, the uPA/uPAR system is a potential target for tumor therapy. We have designed a new generation of uPA-derived synthetic cyclic peptides suited to interfere with the binding of uPA to uPAR and present a new technology involving micro silica particles coated with uPA (SP-uPA) and reacting with recombinant soluble uPAR (suPAR), to rapidly assess the antagonistic potential of uPA-peptides by flow cytofluorometry (FACS). For this, we used silica particles of 10 microm in diameter to which HMW-uPA is coupled using the EDC/NHS method. Soluble, recombinant suPAR was added and the interaction of SP-uPA with suPAR verified by reaction with monoclonal antibody HD13.1 directed to uPAR, followed by a cyan dye (cy5)-labeled antibody directed against mouse IgG. Thereby it was possible to test naturally occurring ligands of uPAR (HMW-uPA, ATF) as well as highly effective, synthetic cyclic uPA-derived peptides (cyclo21,29[D-Cys21Cys29]-UPA21-30, cyclo21,29[D-Cys21Nle28Cys29]-uPA21-30, cyclo21,29[D-Cys(21)2-Nal24Cys29]-uPA21-30, and cyclo21,29[D-Cys21Orn23Thi24Thi25Cys29]-uPA21-30. The results obtained with the noncellular SP-uPA/uPAR system are highly comparable to those obtained with a cellular system involving FITC-uPA and the promyeloid cell line U937 as the source of uPAR.