Small Molecule Inhibitors of Cilia

描述（由申请人提供）：我们开发了一种简单可靠的检测方法来鉴定纤毛组装和功能的小分子抑制剂。纤毛是感觉和运动细胞器，在发育和疾病中起关键作用，包括多囊肾病、脑积水、综合征性肥胖、慢性鼻窦炎、支气管扩张和视网膜变性。小分子调节剂将为研究纤毛动态事件提供关键工具，甚至可能为目前尚无药物可用的纤毛疾病的药理学方法的发展指明道路。我们的实验利用了模式生物衣藻，它利用纤毛游到实验井的底部。因为细胞是绿色的，这导致一个深绿色的颗粒在井中自组织。突变或药物治疗阻断纤毛运动，或导致细胞没有纤毛，阻止这种组织。我们开发了图像分析软件，可以在96孔格式中区分这两种结果。我们已经验证了该分析的性能，发现它的Z值为0.7，板间变异性低，日常重复性高。该分析可以耐受DMSO高达1%，并与高通量机器人方法完全兼容。我们预计，使用这种检测方法的筛选可能为理解纤毛疾病的机制和治疗提供关键的一步。纤毛是蜂窝天线，从人体大多数细胞的表面伸出来，感知周围的环境。这些感觉纤毛在肾脏功能中起着至关重要的作用，纤毛的缺陷导致一种毁灭性的疾病，称为多囊肾病。纤毛还在脂肪代谢和视网膜功能中发挥作用，纤毛缺陷可导致遗传性肥胖和视网膜变性。纤毛也像桨一样在细胞周围移动液体，纤毛运动的缺陷会导致许多疾病症状，包括由于粘液清除不良引起的慢性肺部炎症，以及脑积水（脑积水）。目前还没有治疗疾病的药物，对纤毛如何组装或功能也知之甚少。我们已经开发了一种新的测定方法，用于鉴定可能影响纤毛的化合物。它们不仅将为纤毛如何工作的基础研究提供重要的工具，而且还将作为药物开发工作的先导化合物，旨在治疗目前已知的大量纤毛疾病（纤毛病）。

DESCRIPTION (provided by applicant): We have developed a simple and reliable assay to identify small molecule inhibitors of cilia assembly and function. Cilia are sensory and motile organelles that play critical roles in development and disease, including polycystic kidney disease, hydrocephalus, syndromic obesity, chronic sinusitis, bronchiectasis, and retinal degeneration. Small molecule modulators would provide critical tools for studying dynamic events in cilia, and might even point the way to development of pharmacological approaches to ciliary diseases, for which no drugs are currently available. Our assay takes advantage of the model organism Chlamydomonas, which uses cilia to swim to the bottom of an assay well. Because the cells are green, this causes a dark green pellet to self-organize in the well. Mutations or pharmacological treatments which block ciliary motility, or which cause cells to have no cilia, prevent this organization. We have developed image-analysis software that can distinguish these two results in a 96-well format. We have verified the performance of this assay and found it has a Z- value of 0.7 with low plate-to-plate variability and high day-to-day reproducibility. The assay can tolerate DMSO up to 1% and is completely compatible with high throughput robotic methods. We anticipate that a screen using this assay may provide a critical step forward in understanding the mechanisms and treatment of ciliary diseases. Cilia are cellular antennas which project from the surface of most cells in the human body, and sense the surrounding environment. These sensory cilia play critical roles in kidney function, and defects in cilia lead to a devastating disease called polycystic kidney disease. Cilia also play roles in fat metabolism and retina function, and cilia defects can result in genetic obesity and retinal degeneration. Cilia also act like oars to move fluid around cells, and defects in cilia movement can lead to a number of disease symptoms including chronic lung inflammation due to poor mucus clearance, and to hydrocephalus (water on the brain). Currently there are no drugs available to treat diseases, and little is known about how cilia assemble or function. We have developed a new assay for identifying chemical compounds that can affect cilia. These will not only provide important tools for basic research on how cilia work, they would also serve as lead compounds for drug development efforts aimed at treating the large number of ciliary diseases (ciliopathies) that are now known.