Identification of a novel carbohydrate-mimicking octapeptide from chemical peptide library and characterization as selectin inhibitor.

We found a novel octapeptide (H-YRNWFGRW-NH₂) mimicking sialyl Lewis X (sLe(X)) carbohydrate from a chemical peptide library with anti-sLe(X) monoclonal antibody (MAb) 2H5. The peptide libraries were constructed by Fmoc-based solid-phase methodology using the mix-split method. The octapeptide sequence was determined by the iterative deconvolution method using anti-sLe(X) MAb 2H5. To define the important residues for interaction with anti-sLe(X) MAb 2H5, alanine-scanning analogues of H-YRNWFGRW-NH₂ were synthesized. Substitution of Tyr¹, Trp⁴, Arg⁷ and Trp⁸ to Ala resulted in a marked drop in affinity. This result indicates that aromatic and cationic amino residues have a key role in interacting with anti-sLe(X) MAb 2H5. The binding property of the octapeptide was evaluated with anti-sLe(X) MAb 2H5 and human E-selectin. The octapeptide showed high inhibitory potency (IC₅₀=17.8 nM) for sLe(X) and competitively inhibited the binding of anti-sLe(X) MAb 2H5 in a dose-dependent manner. The octapeptide had high affinity (K(d)=0.168 µM) for E-selectin and this binding was inhibited by sLe(X). These results suggest that octapeptide binds to anti-sLe(X) MAb 2H5 or E-selectin at the sLe(X) binding site and sterically interferes with the recognition of anti-sLe(X) MAb 2H5 or E-selectin with sLe(X). This peptide may be a useful lead compound for an anti-inflammatory agent targeting selectin.