Chemical probes targeting nuclear effector complex of the Hippo signaling pathway

描述(申请人提供)：许多器官，如哺乳动物的肝脏，拥有关于它们最终大小的内在信息，但在动物发育和再生中控制器官大小的分子机制仍然知之甚少。最近河马信号通路的发现为解决这一生物学上长期存在的难题提供了一个前所未有的切入点。最早在果蝇中发现的河马信号通路是一种进化上保守的器官大小调节因子。河马途径的中心是一个从蛋白激酶Hpo/MST到共激活因子yki/yap和DNA结合转录因子SD/tead之间形成的转录因子复合体的激酶级联。与Hippo信号在正常组织动态平衡中的关键作用一致，YAP蛋白在广泛的人类癌症中过度表达或过度激活，这是由于YAP基因座扩增或上游肿瘤抑制因子的遗传/表观遗传失活。YAP的小分子抑制剂不仅将为河马信号的药理调控提供重要的工具，而且在开发治疗由河马信号缺陷引起的人类疾病方面具有巨大的潜力。为此，我们设计并功能验证了一种初步的高通量筛选试验，以发现能够抑制辅活化子yki/yap及其同源转录因子SD/tead相互作用的化学物质。我们已经与NIH化学基因组中心(NCGC)的化学基因组学分部合作，将这一分析小型化到1536平板格式，并成功地与药理活性化合物文库1280(LOPAC1280)进行了中试筛选。在这项拟议的研究中，我们的目标是首先在NCGC筛查中心高通量筛选中实施这种基于靶标的FRET分析，筛选对象是美国国立卫生研究院-分子图书馆小分子资料库(NIH-MLSMR)，收集范围广泛的化学空间。接下来，我们将使用大量预先建立的后续分析来验证主要屏幕点击，以消除假阳性并确定点击集的优先级。最后，我们将测试两个特征良好的热门歌曲 河马通路特异性YAP诱导的小鼠肝增生和肝癌模型。综上所述，我们提出的研究代表了首次大规模筛选活动，以发现针对河马信号通路的先导化合物。

DESCRIPTION (provided by applicant): Many organs such as the mammalian liver possess intrinsic information about their final size, yet the molecular mechanisms that govern organ size in animal development and regeneration remain poorly understood. The recent discovery of the Hippo signaling pathway provides an unprecedented entry point to resolving this long- standing puzzle in biology. First discovered in Drosophila, the Hippo signaling pathway is an evolutionarily conserved regulator of organ size. Central to the Hippo pathway is a kinase cascade leading from the protein kinase Hpo/Mst to a transcription factor complex formed between coactivator Yki/YAP and DNA-binding transcription factor Sd/TEAD. Consistent with the critical role of Hippo signaling in normal tissue homeostasis, the YAP protein is overexpressed or hyperactivated in a wide spectrum of human cancers due to YAP locus amplification or genetic/epigenetic inactivation of upstream tumor suppressors. Small molecule inhibitors of YAP will not only provide important tools for pharmacological manipulation of Hippo signaling, but also bear tremendous potential for developing therapeutic drugs against human diseases caused by defective Hippo signaling. To this end, we have designed and functionally validated a primary high-throughput screen assay to discover chemicals capable of inhibiting the interaction of coactivator Yki/YAP and its cognate transcription factor Sd/TEAD. We have collaborated the Chemical Genomics Branch at NIH Chemical Genomics Center (NCGC) to miniaturize this assay to 1536-plate format and successfully conducted a pilot screen with the Library of Pharmacologically Active Compounds 1280 (LOPAC1280). In this proposed research, we aim to first implement this target-based FRET assay in a high-throughput screen against the National Institute of Health- Molecular Libraries Small Molecule Repository (NIH-MLSMR) collection covering a wide range of chemical space at the NCGC screening center. Next, we will validate the primary screen hits using a plethora of pre- established follow-up assays to remove false positives and prioritize the hit set. Finally we will test the hits in two well-characterized Hippo pathway-specific mouse models of YAP-induced liver hyperplasia and liver cancer. Taken together, our proposed research represents the first-of-its-kind large-scale screening campaign to discover lead compounds targeting the Hippo signaling pathway.