Abstract
We have shown previously that a potent synthetic antagonist of growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domain binding and potent biological activity. To investigate the selectivity of 1 and 3 for Grb2, the biotinylated antagonist 3 was used to immobilize target proteins from cell extracts for subsequent identification by mass spectrometry. Non-specific binding was identified in parallel using a biotinylated analogue that lacked a single critical binding determinant. The mechanism of action of the antagonist was further characterized by immunoprecipitation, immunoblotting, and light microscopy. This approach to defining protein binding antagonist selectivity and molecular basis of action should be widely applicable in drug development.