Chemical probes targeting nuclear effector complex of the Hippo signaling pathway

描述（由申请人提供）：许多器官，如哺乳动物的肝脏，拥有其最终大小的内在信息，然而，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。最近发现的河马信号通路为解决这个长期存在的生物学难题提供了一个前所未有的切入点。首先在果蝇中发现，Hippo信号通路是一种进化上保守的器官大小调节因子。Hippo通路的核心是从蛋白激酶Hpo/Mst到辅激活因子Yki/YAP和dna结合转录因子Sd/TEAD之间形成的转录因子复合物的激酶级联反应。与Hippo信号在正常组织稳态中的关键作用一致，由于YAP基因座扩增或上游肿瘤抑制基因的遗传/表观遗传失活，YAP蛋白在广泛的人类癌症中过度表达或过度激活。YAP的小分子抑制剂不仅将为Hippo信号的药理调控提供重要工具，而且在开发治疗Hippo信号缺陷引起的人类疾病的药物方面具有巨大的潜力。为此，我们设计并功能性验证了一个初级高通量筛选试验，以发现能够抑制辅激活因子Yki/YAP及其同源转录因子Sd/TEAD相互作用的化学物质。我们与NIH化学基因组学中心（NCGC）的化学基因组学分支合作，将该分析缩小到1536个板格式，并成功地通过药理学活性化合物库1280 （LOPAC1280）进行了试点筛选。在这项拟议的研究中，我们的目标是首先在针对NCGC筛选中心覆盖广泛化学空间的美国国立卫生研究院分子文库小分子库（NIH-MLSMR）收集的高通量筛选中实施这种基于靶标的FRET测定。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命中，以消除假阳性并确定命中集的优先级。最后我们将测试命中的两个良好的特征

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.