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

描述（由申请人提供）：蛋白质-蛋白质相互作用构成细胞中几乎所有信号网络的主干。BRCA1是一种1863氨基酸蛋白，其羧基末端结构域（BRCT）有一个磷酸蛋白/肽结合位点（热点）。BACH1和CtIP是结合BRCT热点的两种蛋白，这些相互作用是多种细胞事件的关键，如检查点调节、转录等。BRCT结构域充满了致癌突变，M1775R是阻止BACH1/CtIP与BRCT结合的结构域之一。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M1775R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，我们开发了一种荧光偏振（FP）测定方法，并将其优化为384孔格式，这是进行高通量筛选的理想方法。在这里，我们想提交这个实验来筛选MLSCN小分子库中的化合物，并确定可以用作探针解剖BRCA1信号网络的抑制剂。我们还开发了第二种FP分析，用于探测ZAP70 - N-cbl之间的蛋白质相互作用。该试验可用于对抗筛选BRCA1 FP试验的命中，并确定目标特异性抑制剂。目前在我们的实验室中有一种基于细胞报告的双荧光素酶转录测定方法，可以作为测试机制特异性抑制的二级测定方法。利用这种检测方法的组合，我们建议与MLSCN合作，鉴定并随后优化小分子抑制剂，这些抑制剂可以用作化学探针来询问涉及BRCA1的瞬时蛋白质-蛋白质相互作用。BRCA1是一种1863氨基酸蛋白，具有磷酸化蛋白的结合位点。BRCA1介导的蛋白-蛋白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识别BRCA1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BRCA1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.