Screening of p53-MDM2 interaction inhibitors through genome editing, high-content screening, and surface plasmon resonance

Official URL: http://risc01.sabanciuniv.edu/record=b1817053 (Table of Contents)

The tumor suppressor p53 is the central mediator of cell-cycle arrest, senescence, and apoptosis. p53 protein levels increase upon various cellular stresses to prevent the improper proliferation of cells harboring DNA damage. Under normal conditions, cells keep p53 protein levels suppressed due to its main antagonist, MDM2. This oncogenic protein acts on p53 as an E3 ubiquitin ligase for the polyubiquitination of p53 and its subsequent proteasomal degradation. Activating the p53 pathway is one of the prime targets for novel cancer therapeutics because almost all human cancers have inactivated p53 either by a mutation or by a defect in its regulators, such as the overexpression of MDM2. In this study, we aimed to construct three methods for the screening of novel compounds generated by in silico design and organic synthesis and attempted to inhibit the protein-protein interaction between p53 and MDM2. We generated HCT116 p53-/- MDM2-/- cell lines as a novel assay system through CRISPR/Cas9 genome editing for studying the activity of candidate small molecule compounds targeting the p53-MDM2 interaction. We also constructed a Fluorescent Two-Hybrid (F2H) assay system for highcontent screening of these compounds in real time in living cells and finally a surface plasmon resonance assay for high-throughput screening of these compounds in vitro.