Probing for integrin α_vβ₃ binding of RGD peptides using fluorescence polarization

Integrin α_vβ₃ is an adhesion molecule involved in tumor invasion, angiogenesis, and metastasis. There is substantial interest in developing novel agents that bind to integrin α _vβ₃. Here we report the synthesis and characterization of a fluorescent integrin αvβ₃ probe and its use in a nonradioactive, simple, sensitive fluorescence polarization (FP) assay to quantify binding to integrin αvβ₃. For assay validation, the FP assay was compared to a cell adhesion assay. In the two assays, probe binding to integrin α_vβ₃ showed a similar dependence on probe concentration. The FP assay was successfully applied to measure the binding affinity to integrin α_vβ ₃ of several cyclic peptides containing the Arg-Gly-Asp (RGD) motif. The FP assay we describe here may be appropriate for high-throughput screening for integrin α_vβ₃-binding ligands used for anti-integrin therapy or noninvasive imaging of integrin expression.