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

TY - JOUR

T1 - Peptide-derived antagonists of the urokinase receptor. Affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation

AU - Ploug, M

AU - Østergaard, Søren

AU - Gårdsvoll, H

AU - Kovalski, K

AU - Holst-Hansen, C.

AU - Holm, A.

AU - Ossowski, L.

AU - Danø, K

PY - 2001/10/9

Y1 - 2001/10/9

N2 - 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.

AB - 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.

KW - Animals

KW - Base Sequence

KW - Cell Line

KW - Combinatorial Chemistry Techniques

KW - Cricetinae

KW - DNA Primers

KW - Epitopes

KW - Humans

KW - Mice

KW - Neoplasms

KW - Oligopeptides

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Recombinant Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

KW - Research Support, U.S. Gov't, P.H.S.

M3 - Journal article

C2 - 11580291

SN - 0006-2960

VL - 40

SP - 12157

EP - 12168

JO - Biochemistry

JF - Biochemistry

IS - 40

ER -