Peptide-derived antagonists of the urokinase receptor. Affinity maturationby combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role i n pericellular plasminogen activation. Since proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metasta sis, the uPA-uPAR interaction represents a potential target for therapeutic intervention. By affinity maturation using combinatorial chemistry we have now developed and characterized a 9-mer, linear peptide antagonist of the uPA-uPAR interaction demonstrating specific, high-affinity binding to human uPAR (K-d approximate to 0.4 nM). Studies by surface plasmon resonance rev eal that the off-rate for this receptor-peptide complex is comparable to th at measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagen esis, and its assignment to loop 3 of uPAR domain III (Met(246), His(249), His(251), and Phe(256)) corroborates data previously obtained by photoaffin ity labeling and provides a molecular explanation for the extreme selectivi ty observed for the antagonist toward human compared to mouse, monkey, and hamster uPAR. When human HEp-3 cancer cells were inoculated in the presence of this peptide antagonist, a specific inhibition of cancer cell intravasa tion was observed in a chicken chorioallantoic membrane assay. These data i mply that design of small organic molecules mimicking the binding determina nts of this 9-mer peptide antagonist may have a potential application in co mbination therapy for certain types of cancer.