title: Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist.
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.
authors.affiliation: Urologic Oncology Branch and Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland 20892, USA.
authors.forename: Alessio
authors.initials: A
authors.lastname: Giubellino
authors.forename: Zhen-Dan
authors.initials: ZD
authors.lastname: Shi
authors.forename: Lisa M Miller
authors.initials: LM
authors.lastname: Jenkins
authors.forename: Karen M
authors.initials: KM
authors.lastname: Worthy
authors.forename: Lakshman K
authors.initials: LK
authors.lastname: Bindu
authors.forename: Gagani
authors.initials: G
authors.lastname: Athauda
authors.forename: Benedetta
authors.initials: B
authors.lastname: Peruzzi
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fisher
authors.forename: Ettore
authors.initials: E
authors.lastname: Appella
authors.forename: Terrence R
authors.initials: TR
authors.lastname: Burke
authors.forename: Donald P
authors.initials: DP
authors.lastname: Bottaro
mesh_terms: D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D051380:GRB2 Adaptor Protein / Q000037:antagonists & inhibitors*; D006801:Humans; D015394:Molecular Structure; D013329:Structure-Activity Relationship; D018909:src Homology Domains / Q000187:drug effects*
chemical_list: D051380:GRB2 Adaptor Protein; D001710:Biotin