Chemical probes targeting nuclear effector complex of the Hippo signaling pathway

描述（由申请人提供）：许多器官（如哺乳动物肝脏）具有关于其最终大小的内在信息，但对动物发育和再生中控制器官大小的分子机制仍知之甚少。最近发现的河马信号通路提供了一个前所未有的切入点，以解决这个长期存在的难题在生物学。河马信号通路首先在果蝇中发现，是一种进化上保守的器官大小调节器。Hippo途径的中心是从蛋白激酶Hpo/Mst到共激活因子Yki/雅普和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。与Hippo信号传导在正常组织稳态中的关键作用一致，由于雅普基因座扩增或上游肿瘤抑制因子的遗传/表观遗传失活，雅普蛋白在广谱人类癌症中过表达或过度活化。雅普的小分子抑制剂不仅为Hippo信号的药理学调控提供了重要的工具，而且在开发针对Hippo信号缺陷引起的人类疾病的治疗药物方面具有巨大的潜力。为此，我们设计并功能验证了一个主要的高通量筛选试验，以发现能够抑制辅激活因子Yki/雅普及其同源转录因子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，将该测定法扩展到1536平板格式，并成功地使用药理学活性化合物文库1280（LOPAC 1280）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筛选中心对国家卫生研究所-分子图书馆小分子库（NIH-MLSMR）收集的广泛化学空间进行高通量筛选中实施这种基于靶点的FRET测定。接下来，我们将使用大量预先建立的后续测定来验证初步筛选命中，以去除假阳性并对命中集进行优先级排序。最后，我们将在两个特征良好的 YAP诱导的肝增生和肝癌的Hippo通路特异性小鼠模型。总之，我们提出的研究代表了首次大规模筛选活动，以发现靶向Hippo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.