Development of a scintillation proximity assay for human insulin-like growth factor-binding protein 4 compatible with inhibitor high-throughput screening.
The insulin-like growth factor-binding protein 4 (IGFBP-4), which exists in many different tissues and biological fluids, modulates insulin-like growth factor 1 (IGF-1) bioavailability in part by competitive sequestration and prevention of interaction with cell membrane IGF-1 receptors. Accordingly, small molecules that inhibit the ability of IGF-1 to associate with IGFBP-4 may have clinical utility as regulators of cellular proliferation, survival, and differentiation. Currently, a polyethylene glycol-based precipitation of [(125)I]IGF-1 bound to IGFBP-4 is used to quantify selective IGFBP-4 ligand interactions. We have developed a novel 96-well plate scintillation proximity assay (SPA) for measuring small molecule interactions at IGFBP-4 using a biotinylated form of IGFBP-4 coupled to streptavidin-coated polyvinyltoluene (PVT) SPA microbeads and using [(125)I]IGF-1 as the endogenous ligand. Dose-displacement curves with unlabeled IGF-1 exhibited a mean K(d) value of 0.46 nM. Parallel studies using the nonselective IGFBP inhibitor, NBI-31772, generated a K(i) value of 47 nM. Under optimized conditions, the IGFBP-4 SPA was stable for up to 24h at room temperature and was unaffected by dimethyl sulfoxide (DMSO,<0.5%). This homogeneous binding assay is simple, stable, sensitive, and amenable to automation. The good signal/noise ratio (10:1) and Z' factor (0.7-0.8) make it compatible with high-throughput screening platforms for the identification of IGFBP-4 inhibitors. The IGFBP-4 binding assay may be expanded to other IGFBP members, in biotinylated form, to provide a powerful tool amenable to drug screening and the design of therapeutics to treat a variety of IGF-responsive diseases.
Khawaja
X Z
XZ
Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, USA. khawajx@wyeth.com
D001710:Biotin; D002396:Catechols / Q000494:pharmacology; D002623:Chemistry Techniques, Analytical / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D012588:Scintillation Counting; D019809:Streptavidin
D002396:Catechols; D018974:Insulin-Like Growth Factor Binding Protein 4; D007546:Isoquinolines; D008024:Ligands; C436731:NBI 31772; D007334:Insulin-Like Growth Factor I; D001710:Biotin; D019809:Streptavidin