UBC Theses and Dissertations
Establishing methods to screen novel small molecules targeting insulin-like growth factor/insulin-like growth factor binding protein interaction Alsabban, Abdulrahman Essam
Insulin-like growth factors (IGFs) are important systemic mediators of growth and survival that suppress apoptosis and promote cell cycle progression, angiogenesis and metastatic activities in various cancers by activating IGF-IR tyrosine kinase-mediated signaling. These effects depend on the bioavailability of IGFs, which is regulated by IGF binding proteins (IGFBPs). Increased IGFBP-2 and IGFBP-5 expression observed in castration-resistant prostate cancer is thought to promote tumor progression by enhancing IGF-mediated signaling. IGFBPs have cooperative carboxy-terminal and amino-terminal low and a high affinity IGF binding sites. I hypothesize that blocking the high affinity IGF binding site can affect the bioavailability of IGFs to target tissues and thus be used for treatment of various IGF-responsive diseases including prostate cancer. I initially characterized immunologic reagents capable of being used in sandwich ELISA formats to detect IGF-I and IGFBP-5 and attempted several configurations to establish an IGF-I/IGFBP-5 “bridged” sandwich ELISA platform to measure association and dissociation of IGF-I/IGFBP-5 complex formation. The inability of all bridged ELISA formats tested to measure IGF-I/IGFBP-5 binding, lead me to developed a Bio-Layer Interferometry-based assay that measures IGF-I/ IGFBP-5 binding kinetics that will allow for screening of factors that can affect this intermolecular interaction. I demonstrated that biotinylated IGF-I bound to streptavidin-coated biosensors can be used to measure binding of recombinant IGFBP-5 [2.24 nm shift in optical density (Response)]. I also demonstrated that IGF-I could efficiently disrupt this interaction (0.21 nm shift), while the amino-terminal IGF-I mutant, E3R, exhibits an intermediate competitive activity (1.47 nm shift) and insulin exhibits a low competitive activity (1.83 nm shift). In addition, I demonstrated that IGF-I can competitively disrupted this interaction, resulting in a dissociation rate constant (Kdis 1.5-³ 1/s), In contrast, the amino terminal IGF-I mutant, E3R binds with an intermediate affinity (Kdis 5.6-⁴ 1/s), and buffer free sample results in a (Kdis) of 1.5-⁴ (1/s). These results demonstrate the capacity of this BLI-based assay to differentiate relative competitive activity of compounds that target the high affinity IGF-I binding site of IGFBPs and establish a platform to screen for factors that might be developed as rationale therapeutics to disrupt sequestration of IGF-I by IGFBPs.
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Attribution-NonCommercial-NoDerivatives 4.0 International