title: Peptide-derived antagonists of the urokinase receptor. affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation.
abstract: The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role in pericellular plasminogen activation. Since proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metastasis, 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) approximately 0.4 nM). Studies by surface plasmon resonance reveal that the off-rate for this receptor-peptide complex is comparable to that measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagenesis, and its assignment to loop 3 of uPAR domain III (Met(246), His(249), His(251), and Phe(256)) corroborates data previously obtained by photoaffinity labeling and provides a molecular explanation for the extreme selectivity 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 intravasation was observed in a chicken chorioallantoic membrane assay. These data imply that design of small organic molecules mimicking the binding determinants of this 9-mer peptide antagonist may have a potential application in combination therapy for certain types of cancer.
authors.affiliation: Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark. m-ploug@finsenlab.dk
authors.forename: M
authors.initials: M
authors.lastname: Ploug
authors.forename: S
authors.initials: S
authors.lastname: Østergaard
authors.forename: H
authors.initials: H
authors.lastname: Gårdsvoll
authors.forename: K
authors.initials: K
authors.lastname: Kovalski
authors.forename: C
authors.initials: C
authors.lastname: Holst-Hansen
authors.forename: A
authors.initials: A
authors.lastname: Holm
authors.forename: L
authors.initials: L
authors.lastname: Ossowski
authors.forename: K
authors.initials: K
authors.lastname: Danø
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D020650:Combinatorial Chemistry Techniques; D006224:Cricetinae; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000473:pathology*; D009842:Oligopeptides / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D017931:DNA Primers; D000939:Epitopes; D009842:Oligopeptides; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; C000609091:beta-cyclohexylalanyl-phenylalanyl-seryl-arginyl-tyrosyl-leucyl-tryptophyl-serine