High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.

Abstract

The migration and invasion of cells are necessary for many normal and pathological processes, including tissue remodeling, embryo implantation, angiogenesis, and tumor cell invasion and metastasis (1-4). Recent reports suggest that these processes require an active cell-surface proteolytic cascade (5, 6). Important components of this cascade are the plasminogen activator/plasmin system, as well as the matrix metalloproteinases (6). The requirement for both protease expression and a cell-surface protease binding protein has been demonstrated most clearly in the case of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR) but has also been recently described for type IV collagenase (7,8). It has been shown in human colon and breast carcinomas that urokinase is expressed in stromal, fibroblast-like cells and uPAR is expressed on tumor epithelial cells or macrophages, respectively (9, 10). This suggests that a paracrine relationship between uPA and its receptor occurs in these pathological conditions. The in vitro observation that human tumor cell invasion is proportional to receptor-bound urokinase, not total urokinase synthesis, further supports the hypothesis that cell-surface protease localization is a key for invasion (11,12). Other results show that plasminogen activation is more efficient when both uPA and plasminogen are bound on a cell surface, and that cell-surface plasmin is resistant to inhibition by a2-antiplasmin (7 (23). We report here the identification and characterization of peptide antagonists with nanomolar affinity for the human uPAR by using a 15-mer peptide library. This extension of bacteriophage peptide display to cell-surfaceexpressed proteins expands the utility of the method to a wide variety of biologically interesting targets. MATERIALS AND METHODSReagets and Stains. Bacteriophage library construction and bacteriophage growth and isolation were performed as described by Devlin et al. (15). The Escherichia coli strains H249, a recA, sup0, F' derivative of MM294, and JM103 [F' traD36 proAB+ lacJq lacZAM15 A(pro-lac) supE hsdR endAI sbcBl5 thi-1 strAAi] were used for these experiments. Recombinant DNA manipulations were according to Sambrook et aL (24); electrocompetent E. coil HB101 (Stratagene) were used for subcloning unless otherwise noted. Restriction enzymes were from New England Biolabs; high molecularweight human uPA, plasminogen, and the anti-uPAR monoclonal antibody 3936 were from American Diagnostica (Greenwich, CT). Streptavidin was from Molecular Probes or Sigma, and bovine serum albumin (BSA) was from Sigma. Immulon-2 96-well plates were fom Dynatech. The plasmin substrate S-2251 was from Kabi Pharmacia Diagnostics (Piscataway, NJ). Linear synthetic peptides were prepared on an Applied Biosystems model 430A peptide synthesizer using 9-fluorenylmethoxycarbonyl-based chemistry and were purified by reversed-phase HPLC after trifluoroacetic acid cleavage. Alternatively, peptides were obtained from Chiron Mimotopes (Melbourne, Australia). The cyclic uPA peptide encom...

Cyclo19,31[D-Cys19]-uPA19-31 Is a Potent Competitive Antagonist of the Interaction of Urokinase-Type Plasminogen Activator with Its Receptor (CD87)

“…Using the cyclic disulfide-bridged form of the minimal uPAR-binding region as lead structure (cyclo 19,31 uPA 19-31 ), we have identified cyclo 19,31 ]-uPA 19-31 as -to our knowledge -the most potent competitive uPA-antagonist presently known that displays only a 20-to 40-fold lower uPAR-binding capacity as compared to the natural uPAR ligands uPA or ATF. Cyclo 19,31 ]-uPA 19-31 inhibits binding of uPA to uPAR on human tumor cells with more than 3-fold higher efficiency compared to the lead structure and at least with 10-fold higher efficiency compared to a high affinity uPAR ligand (clone 20 peptide) derived from bacteriophage display (Goodson et al, 1994;Bürgle et al, 1997). Furthermore, cyclo 19,31 ]-uPA 19-31 efficiently displaces uPAR-bound uPA from the cell surface of human cells and inhibits uPA-mediated, cell-associated plasminogen activation and fibrin degradation.…”

“…Other types of peptide antagonists of uPA/uPAR-interaction, which show no obvious similarity to uPA [19][20][21][22][23][24][25][26][27][28][29][30][31] , have been identified by a bacteriophage peptide display approach (Goodson et al, 1994). All of the reactive peptides (with the clone 20 peptide being most active) harbor relatively short common motifs, such as LWXXY and FXXYLW, which may represent a variation of the motif 24 YFXXIXW 30 within the uPA molecule (only the critical hydrophobic amino acids for uPAR-binding are depicted) (Magdolen et al, 1996;Bürgle et al, 1997).…”

Role of plasminogen activator-plasmin system in tumor angiogenesis

“…Peptides are predicted to lack stability due to susceptibility to exoproteinase degradation in the plasma. High-affinity peptide ligands of uPAR that have been identified by phage display [112,113] may circumvent some of the pharmacological problems typically associated with peptide therapy. Alternatively, anti-uPAR antibodies [114] or non-peptidic small-molecule antagonists of uPA binding have been described [100].…”

Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

“…And lots of antagonists were identified from phage displayed peptide libraries, such as urokinase receptor antagonists and the human type I interleukin (IL)-1 receptor antagonist [14][15][16][17]. However, there are some difficulties for membrane receptors to be purified and maintain the natural conformation.…”