Probing for Integrin αvβ3Binding of RGD Peptides Using Fluorescence Polarization

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Abstract
Integrin αvβ3 is an adhesion molecule involved in tumor invasion, angiogenesis, and metastasis. There is substantial interest in developing novel agents that bind to integrin αvβ3. 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 αvβ3 showed a similar dependence on probe concentration. The FP assay was successfully applied to measure the binding affinity to integrin αvβ3 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 αvβ3-binding ligands used for anti-integrin therapy or noninvasive imaging of integrin expression.