Phage Libraries Displaying Cyclic Peptides with Different Ring Sizes: Ligand Specificities of the RGD-Directed Integrins

We have isolated selective ligands to the cell surface receptors of fibronectin (₅₁ integrin), vitronectin (_v3 and _v3 integrins) and flbrinogen (_IIb3 integrin) from phage libraries expressing cyclic peptides. A mixture of libraries was used that express a series of peptides flanked by a cysteine residue on each side (CX₅C, CX₆C, CX₇C) or only on one side (CX₉) of the insert. A majority of the integrin-binding sequences derived from the CX, library contained another cysteine, indicating preferential selection of conformationally constrained cyclic peptides. Each of the four integrins studied primarily selected RGD-containing phage sequences but favored different ring sizes and different flanking residues around the RGD motif. A cyclic peptide ACRGDGWCG was synthesized based on a phage sequence that bound particularly avidly to the ₅₁ integrin. This peptide inhibited cell attachment to fibronectin at about 5-fold lower concentrations than the most potent cyclic peptides described earlier. The most interesting structure appeared to contain two disulphide bonds. One such peptide, ACDCRGDCFCG, was synthetized and shown to be at least 20-fold more potent inhibitor of _v5- and _v3-mediated cell attachment to vitronectin than similar peptides with a single disulphide bond and 200-fold more potent than commonly used linear RGD peptides. These results emphasize the importance of conformational restriction as a means of improving the potency of integrin-binding peptides and point to a new way of designing effective peptides by resticting the peptide conformation with more than one cyclizing bond.

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