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

描述（由申请人提供）：蛋白质-蛋白质相互作用形成细胞中几乎所有信号网络的骨架。BRCA 1是一种1863个氨基酸的蛋白质，在其羧基末端结构域（BRCT）中具有磷蛋白/肽结合位点（热点）。BACH 1和CtIP是两种与BRCT热点结合的蛋白质，这些相互作用是各种细胞事件的关键，如检查点调节，转录等。BRCT结构域充满了致癌突变，M1775 R是阻止BACH 1/CtIP与BRCT结合的蛋白质。基于临床前和回顾性临床研究，有一种新兴的理论表明BRCT突变使癌细胞对基于DNA损伤的化疗药物敏感。我们假设靶向BRCT热点并在功能上模拟M1775 R突变的可逆抑制剂将使细胞对基于DNA损伤的化疗药物敏感。为了验证这一假设，我们开发了荧光偏振（FP）测定法，并将其优化为384孔格式，这是进行高通量筛选的理想选择。在这里，我们想提交这种检测方法来筛选MLSCN小分子库中的化合物，并鉴定可用作探针来分析BRCA 1信号网络的抑制剂。我们还开发了第二种FP测定法，其探测ZAP 70- N-cbl之间的蛋白质-蛋白质相互作用。该测定可用于反筛选来自BRCA 1 FP测定的命中并鉴定靶特异性抑制剂。我们实验室目前可用的基于细胞的报告基因的双荧光素酶转录测定可用作检测机制特异性抑制的二级测定。使用这种检测组合，我们建议与MLSCN合作，以识别并随后优化可用作化学探针的小分子抑制剂，以询问涉及BRCA 1的瞬时蛋白质-蛋白质相互作用。BRCA 1是一种由1863个氨基酸组成的蛋白质，具有磷酸化蛋白质的结合位点。BRCA 1介导的蛋白质-蛋白质相互作用是复杂信号网络的一部分。本申请描述了一组可用于鉴定BRCA 1的小分子抑制剂的生物测定。这些抑制剂是有价值的化学探针，可用于更好地了解BRCA 1突变患者的肿瘤发生。

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.