Multivalency is a powerful strategy for achieving high-affinity molecular binding of compounds to increase their therapeutic potency or imaging potential. In our study, multivalent non-peptide integrin αvβ3 antagonists (IA) were designed for antitumor therapy. Docking and molecular dynamics were employed to explore the binding modes of IA monomer, dimer, and trimer. In silico, one IA unit binds tightly in the active site with similar pose to native ligand RGD and other parts of dimer and trimer contribute extra binding affinities by interacting with residues in vicinity of the original site. In vitro studies demonstrated that increasing valency results in increasing antiproliferative and antiorganizational effects against endothelial cells (HUVECs), and a much weaker effect on melanoma B16F10 cells. The antitumor efficacies of the IA multivalent compounds were evaluated in subcutaneous B16F10 melanoma tumor-bearing mice. At 30 mg/kg dose, the mean masses of tumors harvested 18 days after inoculation were significantly reduced (p<10(-7)) by 36±9%, 49±8%, and 71±7% for the IA monomer, dimer, and trimer groups, relative to control. The importance of multivalency was demonstrated to be highly significant beyond the additive effect of the extra pharmacological sites (p=0.00011). These results suggest that the major target of these anti-αvβ3 compounds is the neovasculature rather than the cancer cells, and the success of a multivalent strategy depends on the details of the components and linker. This is the first integrin αvβ3 multivalent ligand showing clear enhancement in antitumor effectiveness.
