Structure/activity studies of anti-inflammatory peptides based on a conserved peptide region of the lectin domain of E-, L- and P-selectin

Abstract

Previously, it was established that the peptide YYWIGIRK-NH2 inhibits both myeloid cell adhesion to selectins in vitro and neutrophil influx into inflammatory sites in vivo (Briggs et al., 1995). Initial structure/activity studies revealed that at least one Y residue at the N-terminus of the peptide was essential for these bioactivities but that the C-terminal K residue was unnecessary for inhibitory activity. We have now synthesized a new series of peptides which contain single residue substitutions at each position of the reference peptide, YYWIGIR-NH2, and have tested these peptides for inhibitory activity in a selectin cell binding assay. In addition, peptides containing single D-amino acids at selected positions, or an all D-configured reference peptide sequence, or the retro-inverso version (rigiwyy-NH2) of the reference peptide sequence have also been analyzed for inhibitory activity in the same assays. Finally, the ability of the reference peptide and a specifically designed control sequence (YY(AIB)IGIR-NH2) to discriminate between potential synthetic saccharide ligands, including sialyl-Lewis x, Lewis x, and sialyl-N-acetyl-lactosamine, was investigated using isothermal titration calorimetry. The results of these studies demonstrate that whereas many single amino acid substitutions are tolerated in the peptide without complete loss of inhibitory activity, substitution at some positions (e.g., the W residue) results in relatively inactive compounds, clearly pointing to the importance of these residues in making critical contacts with the appropriate saccharide ligand. Titration calorimetry revealed that the reference peptide does not discriminate between Lewis x or sialyl-Lewis x in vitro, but binds these saccharides with nearly 40-fold higher affinity (KD 25 microM) than the nonfucosylated trisaccharide, sialyl-N-acetyl-lactosamine. We can infer from these studies that the presence of a sialyl group per se, is not a requisite for complex formation between the reference peptide and its saccharide ligand. Substitution of single D-amino acid residues at various positions in the reference peptide sequence reduces or eliminates all inhibitory properties. However, the all D-configured peptide or the retro-inverso peptide sequence have greater activity than the all L-configured reference peptide in the in vitro biological assays, and each was an effective inhibitor of neutrophil infiltration in a thioglycolate-induced mouse peritonitis model. These results, combined with the results of titration, allow us to conclude that binding between the reference peptide and its saccharide ligand, which affords its inhibitory properties, is mediated by the presence of a contiguous, nonpolar surface, or face, presented at the N-terminus of the reference peptide, likely encompassing the sequence YYWI. Furthermore, the W plays a critical role in binding, probably through formation of an essential hydrogen bond with a suitably juxtaposed group carried on the saccharide ligand.

Carbohydrate Chain of Ganglioside GM1 as a Ligand: Identification of the Binding Strategies of Three 15 mer Peptides and Their Divergence from the Binding Modes of Growth‐Regulatory Galectin‐1 and Cholera Toxin

“…Notably, the hydrophobic contact area of this protein is confined to the sialic acid's N‐acetyl group, which offers another option for affinity enhancements in both cases. The apparently enormous potential of a contiguous non‐polar sequence stretch for conferring affinity to a short peptide had been deduced with the selectin‐based heptamer YYWIGIR, although the binding of the full‐length E‐ and P‐selectins to the sialyl Lewis x determinant is primarily electrostatic in nature, in line with its binding kinetics required for a molecular braking mechanism 53. 54 The small plant lectins hevein and pseudohevein also illustrate the extent of stacking to generate affinity 55.…”

Selectin−Carbohydrate Interactions: From Natural Ligands to Designed Mimics

“…Martens and colleagues at Affymax applied phage display to find peptide ligands that bind to E-selectin and prevent neutrophil adhesion (Table ) …”

Therapeutic potential of a novel synthetic selectin blocker, OJ‐R9188, in allergic dermatitis

“…In the early stages of thioglycollate‐induced peritoneal inflammation in mice, anti‐P‐selectin and anti‐L‐selectin, but not anti‐E‐selectin, antibodies prevent neutrophil infiltration into the peritoneal cavity. These results indicate that P‐ and L‐selectins are involved in the neutrophil extravasation in response to thioglycollate (Arbones et al ., 1994; Mayadas et al ., 1993; Labow et al ., 1994; Briggs et al ., 1996; Bosse & Vestweber, 1994). The intravenous injection of OJ‐R9188 significantly reduced thioglycollate‐induced neutrophil infiltration in the peritoneal cavity.…”