Effect of multimerization of a linear Arg-Gly-Asp peptide on integrin binding affinity and specificity

Multivalent interactions are frequently used to enhance ligand-receptor binding affinity. In this study, mono-, di- and trimeric Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Tyr (AVTGRGDSY) peptides, labeled with (125)I or Cy5.5, were compared in vitro and in vivo. Using human embryonic kidney HEK293 (naturally alpha(V)-positive and beta(3)-negative), HEK293(beta(1)) (beta(1)-transfected and alpha(V)beta(3)-negative), HEK293(beta(3)) (beta(3)-transfected and strongly alpha(V)beta(3)-positive), and human glioblastoma U87MG (naturally alpha(V)beta(3)-positive) cell lines we evaluated their binding affinity and specificity. In vitro, the monomeric AVTGRGDSY showed specific binding to both HEK293(beta(1)) and HEK293(beta(3)) cells. Multimerization resulted in no change toward HEK293 cells, diminished binding with HEK293(beta(1)) cells, but substantially enhanced binding with alpha(V)beta(3)-positive HEK293(beta(3)) and U87MG cells. Moreover, multimeric AVTGRGDSY peptides were found to be nearly comparable to the same molar concentration of a well-known alpha(V)beta(3)-specific cyclo(RGDfV) (c(RGDfV)) peptide in specificity and affinity for targeting alpha(V)beta(3) integrin. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric analogue, the Cy5.5-labeled dimeric AVTGRGDSY peptide produced markedly enhanced tumor-to-background contrast in HEK293(beta(3)) tumor-bearing mice than in HEK293(beta(1)) tumor-bearing mice. In conclusion, the present study showed the difference of monomeric and multimeric linear Arg-Gly-Asp (RGD)-containing compound in integrin selectivity and affinity. Our data provide useful information for the design of novel RGD peptides.