HTS to Identify Small Molecule Inhibitors of Phospho-Specific Protein-Protein Int

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DESCRIPTION (provided by applicant): Protein-protein interactions form the backbone of nearly all the signaling networks in the cells. BRCA1 is an 1863 amino acid protein that has a phosphoprotein / peptide binding site (hot spot) in its carboxy terminus domains (BRCT). BACH1 and CtIP are two proteins that bind to the BRCT hot spot and these interactions are key for a variety of cellular events such as checkpoint regulation, transcription etc. BRCT domains are riddled with cancer causing mutations and M1775R is one which prevents BACH1/CtIP binding to BRCT. Based on preclinical and retrospective clinical studies there is an emerging theory that suggests that BRCT mutations sensitize cancer cells to DNA damage based chemotherapeutics. We hypothesize that reversible inhibitors that target the hot spot on BRCT and functionally mimic the M1775R mutation will sensitize cells to DNA damage based chemotherapeutics. To test this hypothesis we developed a fluorescence polarization (FP) assay and optimized it to a 384-well format which is ideal to carry out high throughput screening. Here we would like to submit this assay to screen the compounds in the small molecule repository at the MLSCN and identify inhibitors that can be used as probes to dissect the BRCA1 signaling network. We also have developed a second FP assay that probes the protein-protein interaction between ZAP70 - N-cbl. This assay can be used as to counter screen the hits from the BRCA1 FP assay and identify target specific inhibitors. A cell based reporter based dual luciferase transcription assay that is currently available in our laboratory can be used as a secondary assay to test for mechanism specific inhibition. Using this combination of assays we propose to work with the MLSCN to identify and subsequently optimize small molecule inhibitors that can be used as chemical probes to interrogate the transient protein-protein interactions involving BRCA1. BRCA1 is and 1863 amino acid protein that has a binding site for phosphorylated proteins. The BRCA1 mediated protein-protein interactions is part of a complex signaling network. This application describes a set of biological assays that can be used to identify small molecule inhibitors of BRCA1. These inhibitors are valuable chemical probes that can be used to better understand tumorigenesis in patients with BRCA1 mutations.