Development of a scintillation proximity assay for human insulin-like growth factor-binding protein 4 compatible with inhibitor high-throughput screening

• Published in: Analytical biochemistry Vol. 366; no. 1; pp. 80 - 86
• Main Author: Khawaja, X.Z.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2007
• Subjects: [ 125I]IGF-1; Biotin; Catechols - pharmacology; Chemistry Techniques, Analytical - methods; Drug Evaluation, Preclinical - methods; High-throughput screening; Humans; IGF-1; IGFBP-4; In Vitro Techniques; Insulin-Like Growth Factor Binding Protein 4 - analysis; Insulin-Like Growth Factor Binding Protein 4 - antagonists & inhibitors; Insulin-Like Growth Factor Binding Protein 4 - metabolism; Insulin-Like Growth Factor I - metabolism; Isoquinolines - pharmacology; Kinetics; Ligands; NBI-31772; Receptor binding assay; Scintillation Counting; Scintillation proximity assay; Streptavidin; NBI-31772; Scintillation proximity assay; IGFBP-4; Receptor binding assay; High-throughput screening; IGF-1; [ 125I]IGF-1
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.03.011

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 [ 125I]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 [ 125I]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 24 h 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.