Specific inhibition of P-selectin-mediated cell adhesion by phage display-derived peptide antagonists.
P-selectin is a leukocyte adhesion receptor expressed on activated vascular endothelium and platelets that mediates leukocyte rolling and attachment. Because P-selectin is critically involved in inflammation, we used phage display libraries to identify P-selectin-specific peptides that might interfere with its proinflammatory function. Isolated phage contained a highly conserved amino acid motif. Synthetic peptides showed calcium-dependent binding to P-selectin, with high selectivity over E-selectin and L-selectin. The peptides completely antagonized adhesion of monocyte-derived HL60 cells to P-selectin and increased their rolling velocities in flow chamber experiments. Peptide truncation and alanine-scanning studies indicated that an EWVDV (single-letter amino acid codes) consensus motif sufficed for effective inhibition. Intriguingly, the apparent avidity of the peptides was increased 200-fold when presented in a tetrameric form (2 microM versus 10 nM), which is consistent with the proposed divalent interaction of P-selectin glycoprotein ligand 1 (PSGL-1) with P-selectin. As the EWVDV peptides inhibit the binding of an established glycoside ligand for P-selectin (sulfated Lewis A), it is conceivable that EWVDV interacts with or in close proximity to the actual carbohydrate recognition domain of P-selectin, without being a direct structural mimic of sialyl Lewis(x). These ligands are among the most potent antagonists of P-selectin yet designed. Their high affinity, selectivity, and accessible synthesis provide a promising entry to the development of new anti-inflammatory therapeutics and might be a powerful tool to provide important information on the binding site of P-selectin.
Molenaar
Tom J M
TJ
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Appeldoorn
Chantal C M
CC
de Haas
Sonja A M
SA
Michon
Ingrid N
IN
Bonnefoy
Arnaud
A
Hoylaerts
Marc F
MF
Pannekoek
Hans
H
van Berkel
Theo J C
TJ
Kuiper
Johan
J
Biessen
Erik A L
EA
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects*; D019281:Dimerization; D018922:HL-60 Cells; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism; D009842:Oligopeptides / Q000037:antagonists & inhibitors* / Q000737:chemistry; D019007:P-Selectin / Q000187:drug effects* / Q000378:metabolism / Q000494:pharmacology; D019151:Peptide Library; D011485:Protein Binding / Q000187:drug effects
D008024:Ligands; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D019007:P-Selectin; C084882:P-selectin ligand protein; D019151:Peptide Library; D002118:Calcium