HIGH-AFFINITY UROKINASE RECEPTOR ANTAGONISTS IDENTIFIED WITH BACTERIOPHAGE PEPTIDE DISPLAY

Affinity selection of a 15-mer random peptide library displayed on bac teriophage M13 has been used to identify potent ligands for the human urokinase receptor, a key mediator of tumor cell invasion. A family of receptor binding bacteriophage ligands was obtained by sequentially a nd alternately selecting the peptide library on COS-7 monkey kidney ce lls and baculovirus-infected Sf9 insect cells overexpressing the human urokinase receptor. Nineteen peptides encoded by the random DNA regio ns of the selected bacteriophage were synthesized and tested in a urok inase receptor binding assay, where they competed with the labeled N-t erminal fragment of urokinase with IC50 values ranging from 10 nM to 1 0 mu M. All of the isolated peptides were linear and showed two relati vely short conserved subsequences: LWXXAr (Ar = Y, W, F, or H) and XFX XYLW, neither of which is found in urokinase or its receptor. Competit ion experiments demonstrated that the most potent peptide, clone 20, p revented binding of bacteriophage displaying the urokinase receptor bi nding sequence (urokinase residues 13-32). In addition, this peptide b locked other apparently unrelated receptor binding bacteriophage, sugg esting overlapping receptor interaction sites for all of these sequenc es. These results provide a demonstration of bacteriophage display ide ntifying peptide ligands for a receptor expressed on cells and yield l eads for the development of urokinase receptor antagonists.