Rational Optimization of a Short Human P-selectin-binding Peptide Leads to Nanomolar Affinity Antagonists

Abstract

P-selectin plays an important role in the development of various diseases, including atherosclerosis and thrombosis. In our laboratory we recently identified a number of specific human P-selectin-binding peptides containing a Glu-Trp-Val-Asp-Val consensus motif, displaying a low micromolar affinity for P-selectin (IC 50 ‫؍‬ 2 M). In search of more potent antagonists for P-selectin, we have optimized the EWVDV pentapeptide core motif via a two-step combinatorial chemistry approach. A dedicated library of peptide derivatives was generated by introducing seven substituents at the N and C termini of the motif. In particular, pentapeptides with gallic acid or 1,3,5-benzenetricarboxylic acid substituents at the N terminus proved to be considerably more potent inhibitors of P-selectin binding than the parental peptide. After removal of the N-terminal glutamic acid from the core sequence, which appeared to be replaceable by a carboxamide function without loss of affinity, a second library was synthesized to map the chemical moieties within the gallic acid or 1,3,5-benzenetricarboxyl acid groups responsible for the enhanced P-selectin binding. Moreover, by varying the length and rigidity of the connective spacer, we have further optimized the spatial orientation of the N-terminal substituent. The combined use of phage display and subsequent combinatorial chemistry led to the design of a number of gallic acidcontaining peptides with low nanomolar affinity for P-selectin both under static and dynamic conditions (IC 50 ‫؍‬ 15.4 nM). These small synthetic antagonists, which are equally as potent as the natural ligand Pselectin glycoprotein ligand-1, are promising leads in anti-atherothrombotic therapy.P-selectin, a cell adhesion molecule involved in the initial attachment and "rolling" of leukocytes across the inflamed vessel wall (1-4), plays a key role in atherosclerosis. In fact, P-selectin deficiency in mice has been shown to reduce atherosclerotic lesion formation (5). Also, P-selectin activation induces hypercoagulance of platelets and mediates plateletmonocyte aggregation and has thus been associated with thrombosis (6 -9). Therefore, intervention in P-selectin-mediated processes is an attractive therapeutic entry.The endogenous ligand for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1), 1 a 220-kDa disulfide-linked homodimeric sialomucin (10). P-selectin binding to PSGL-1 proceeds via a short N-terminal amino acid sequence of the latter, containing three sulfated tyrosines and a sialyl Lewis X (sLeX, Neu5Ac␣2-3Gal␤1-4[Fuc␣1-3]GlcNAc) moiety (11-14). Most P-selectin antagonists are carbohydrate derivatives of the sLeX structure (15). However, as compared with PSGL-1, these glycosides are relatively poor and unselective P-selectin inhibitors, because binding to the other selectin family members (Eand L-selectin) is equally affected.In our lab we recently identified, through the use of phage display, a number of human P-selectin-binding peptides containing an EWVDV pentapeptide consensus motif (16). Binding of t...

Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes

“…To neutralize P-selectin in vivo, we used GA, a well-known polyphenol acting as a selective antagonist of P-selectin, but not of E-selectin. 34,35,46 As expected (because P-selectin is poorly expressed on quiescent ECs), GA did not alter the interactions of leukocytes with resting endothelium in either genotype ( Figure 2D). However, when the endothelium was selectively activated by topical administration of A23187, GA inhibited the interactions of leukocytes with ECs in WT mice, notably, to the same level as in Gas6 Ϫ/Ϫ mice, whereas GA was ineffective in Gas6 Ϫ/Ϫ mice ( Figure 2D).…”

“…Gallic acid (GA; 3.4 mg/kg per hour), previously shown to selectively inhibit P-selectin, was continuously administered intravenously via an infusion pump, as described previously. 34,35 The mesentery of recipient mice was externalized, and mesenteric venules were exposed on the table of an epifluorescence microscope for direct visualization under videomicroscopy. Local EC activation was induced via topical application of the calcium ionophore A23187 (10 L at 30 M).…”

Pharmacological Inhibition of Platelet-Tumor Cell Cross-Talk Prevents Platelet-Induced Overexpression of Cyclooxygenase-2 in HT29 Human Colon Carcinoma Cells

“…3E). In contrast, experiments performed with a P-selectin antagonist (Appeldoorn et al, 2003) led us to exclude its role in platelet-HT29 cell interactions (Fig. 3F).…”

Gallic Acid Antagonizes P-Selectin–Mediated Platelet–Leukocyte Interactions

“…19,34 Previously, we reported that derivatization of specific human P-selectin-binding peptides with GA led to a dramatically enhanced affinity of the peptide toward P-selectin. 17 This led to the presumption that GA by itself may specifically interact with P-selectin. Indeed, the polyphenol was found to inhibit the binding of 2 different selectin ligands, TM11-PO 22 and biotin-PAA-Le a -SO 3 H, 23 to P-selectin at micromolar affinity.…”

“…Interestingly, we previously found that derivatization of peptide antagonists for human P-selectin with GA enhanced their affinity for P-selectin Ͼ500-fold. 17 P-selectin is an adhesion molecule that is intrincally implicated in atherothrombosis by mediating leukocyte-endothelium, leukocyte-platelet, and plateletplatelet interactions. 18,19 The absence of P-selectin was found to result in reduced atherosclerotic lesion development and neointimal growth.…”