High-throughput imaging of Hedgehog pathway components at the primary cilium

描述（由申请人提供）：Sonic hedgehog（Shh）通路参与各种形式的癌症，包括脑、皮肤、胰腺、卵巢和胃肠道肿瘤。靶向该通路的药物对肿瘤学有很大的希望，但目前这一代的Shh通路抑制剂导致耐药性的出现。大多数抑制Shh途径的已知化合物是通过基于人工靶荧光素酶构建体的表达的高通量筛选发现的。然而，这种方法有一些主要的缺点，并产生大量的错误命中。在这里，我们建议开发一种新的高通量筛选方法的基础上亚细胞定位的Shh通路组件的细胞器称为初级纤毛。我们的方法不仅比目前使用的筛选方法更稳健，而且筛选过程中产生的高内容数据可用于探测Shh信号传导和纤毛功能的分子机制。我们将使用这种新开发的高通量筛选试验来发现新的Shh抑制剂，以防止癌细胞逃避治疗。 公共卫生相关性：新药的发现是通过筛选大量化合物来实现的，这些化合物能够阻止仅在疾病状态（如癌症）中发现的特定生化事件。某些形式的癌症，如儿童脑肿瘤成神经管细胞瘤，皮肤癌，以及胰腺，结肠和卵巢的肿瘤，其特征在于蛋白质向细胞中称为初级纤毛的微小结构的运动增加。我们将开发一种自动监测蛋白质运动到初级纤毛的方法，这将使我们能够测试数十万种化学分子阻断这种运输现象的能力，希望发现潜在的新一代抗癌药物。

DESCRIPTION (provided by applicant): The Sonic hedgehog (Shh) pathway is involved in various forms of cancer, including brain, skin, pancreatic, ovarian, and gastrointestinal neoplasms. Drugs targeting this pathway hold great promise for oncology, but the current generation of Shh pathway inhibitors cause emergence of drug resistance. Most of the known compounds that inhibit the Shh pathway were discovered by high-throughput screening based on the expression of an artificial target luciferase construct. This method, however, has some major drawbacks and yields a large number of false hits. Here, we propose to develop a novel high-throughput screening method based on subcellular localization of Shh pathway components to an organelle called the primary cilium. Our method will not only be more robust than the currently used screening methods, but the high-content data generated in the course of screening can be used to probe molecular mechanisms of Shh signaling and ciliary function. We will use this newly developed high-throughput screening assay to discover new Shh inhibitors that will prevent cancer cells from evading treatment. PUBLIC HEALTH RELEVANCE: The discovery of new pharmaceuticals is accomplished by screening vast collections of chemical compounds for their ability to block specific biochemical events found only in disease states, such as cancer. Certain forms of cancer, such as a childhood brain tumor medulloblastoma, skin cancer, and tumors of the pancreas, colon, and ovaries, are characterized by increased movement of proteins to a tiny structure in the cell called the primary cilium. We will develop a method of automatic monitoring protein movement to the primary cilium, which will enable us to test hundreds of thousands of chemical molecules for their ability to block this transport phenomenon in hopes of discovering a potential new generation of anti-cancer drugs.