A high content screen dissecting ciliogenesis and oncogenic Hedgehog signaling

 描述（由申请人提供）：本提案旨在鉴定和表征抑制纤毛Hedgehog（Hh）信号传导的药物样分子。细胞通过分泌信号（如Hh蛋白）进行交流来协调它们的行为。与其他细胞间信号不同，哺乳动物Hh蛋白通过初级纤毛（许多细胞表面上基于微管的突起）转导。Reiter实验室发现，Hh通路的一个中心成分Smoothened（Smo）移动到初级纤毛以响应Hh刺激，在那里它激活下游通路。在胚胎发育和肿瘤发生中，纤毛对于Hh信号传导是必不可少的。Smo的激活突变导致基底细胞癌（BCC），这是美国最常见的癌症，和儿童最常见的实体癌髓母细胞瘤。Smo拮抗剂最近被批准用于临床使用，尽管癌症可以很快产生耐药性。通过互补机制起作用的Smo拮抗剂可以防止Hh相关癌症中出现耐药性。为了鉴定新的Hh通路拮抗剂并深入了解纤毛功能，Reiter和Arkin实验室完成了Smo运动到纤毛的抑制剂的试点筛选。该筛选确定了新的抑制剂，其中一些 其中一些通过抑制Smo易位阻断BCC细胞生长，并且其中一些阻断纤毛发生。研究这些抑制剂的作用机制揭示了睫状体Hh信号传导的以前未知的方面。这个项目将扩大筛选，确定抑制剂作用的分子机制，并了解纤毛信号。具体而言，我们将：1）筛选内部和化学生物学联盟文库，大大扩展已评估的化学空间，2）执行二次筛选以有效地鉴定通过先前未描述的机制起作用的抑制剂，3）使用新鉴定的拮抗剂，连同独特的遗传工具，以揭示纤毛发生和Smo易位的未识别步骤。尽管它们对发育和疾病都很重要，但纤毛发生和Hh信号转导的机制仍不清楚。这项研究将提供工具来揭示细胞如何通过纤毛进行交流。这些化合物还可以为新型化疗药物提供线索，这对于预防Hh通路相关癌症中出现耐药性和复发至关重要。

 DESCRIPTION (provided by applicant): This proposal seeks to identify and characterize drug-like molecules that inhibit ciliary Hedgehog (Hh) signaling. Cells orchestrate their behaviors by communicating through secreted signals such as Hh proteins. Unlike other intercellular signals, mammalian Hh proteins are transduced through primary cilia, microtubule-based projections on the surface of many cells. The Reiter lab discovered that a central component of the Hh pathway, Smoothened (Smo), moves to the primary cilium in response to Hh stimulation where it activates the downstream pathway. The cilium is essential for Hh signaling both in embryonic development and in oncogenesis. Activating mutations in Smo cause basal cell carcinoma (BCC), the most common cancer in the U.S., and medulloblastoma, the most common solid cancer in children. A Smo antagonist was recently approved for clinical use, although cancers can become resistant quickly. Smo antagonists that act through complementary mechanisms may prevent the emergence of resistance in Hh-associated cancers. To identify novel Hh pathway antagonists and gain insight into ciliary function, the Reiter and Arkin labs completed a pilot screen for inhibitors of Smo movement to cilia. This screen identified novel inhibitors, some of which block BCC cell growth by inhibiting Smo translocation and some of which block ciliogenesis. Investigating the mechanisms by which these inhibitors act revealed previously unknown aspects of ciliary Hh signaling. This project will expand the screen, identify the molecular mechanisms underlying inhibitor action, and understand ciliary signaling. Specifically, we will: 1) screen in-house and Chemical Biology Consortium libraries, substantially expanding the chemical space that has been assessed, 2) execute secondary screens to efficiently identify inhibitors that act through previously undescribed mechanisms, 3) use newly identified antagonists, together with unique genetic tools, to uncover unrecognized steps of ciliogenesis and Smo translocation. Despite their importance to both development and disease, the mechanisms underlying ciliogenesis and Hh signaling remain unclear. The proposed investigation will provide tools to reveal how cells communicate through cilia. These compounds may also provide leads for novel chemotherapeutics, critical for preventing the emergence of resistance and relapse in Hh pathway-associated cancers.