Proligands with protease-regulated binding activity identified from cell-displayed prodomain libraries.
A general method was developed for the discovery of protease-activated binding ligands, or proligands, from combinatorial prodomain libraries displayed on the surface of E. coli. Peptide libraries of candidate prodomains were fused with a matrix metalloprotease-2 substrate linker to a vascular endothelial growth factor-binding peptide and sorted using a two-stage flow cytometry screening procedure to isolate proligands that required protease treatment for binding activity. Prodomains that imparted protease-mediated switching activity were identified after three sorting cycles using two unique library design strategies. The best performing proligand exhibited a 100-fold improvement in apparent binding affinity after exposure to protease. This method may prove useful for developing therapeutic and diagnostic ligands with improved systemic targeting specificity.
Thomas
Jerry M
JM
Department of Chemical Engineering, University of California, Santa Barbara, 93106, USA.
Daugherty
Patrick S
PS
D003001:Cloning, Molecular; D020650:Combinatorial Chemistry Techniques; D004926:Escherichia coli; D006801:Humans; D008024:Ligands; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000494:pharmacology*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D042461:Vascular Endothelial Growth Factor A; D010447:Peptide Hydrolases; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2