Duncan Mara C, Ho David G, Huang Jing, Jung Michael E, Payne Gregory S
Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Proc Natl Acad Sci U S A. 2007 Apr 10;104(15):6235-40. doi: 10.1073/pnas.0607773104. Epub 2007 Apr 2.
Small molecule inhibitors provide powerful tools to characterize highly dynamic and complex eukaryotic cell pathways such as those mediating membrane traffic. However, a lack of easy and generalizable assays has constrained identification of novel inhibitors despite availability of diverse chemical libraries. Here, we report a facile growth-based strategy in yeast to screen for pathway-specific inhibitors. The approach uses well characterized synthetic genetic growth defects to guide design of cells genetically sensitized for inhibition of chosen pathways. With this strategy, we identified a family of piperazinyl phenylethanone compounds as inhibitors of traffic between the trans-Golgi network (TGN) and endosomes that depends on the clathrin adaptor complex AP-1. The compounds did not significantly alter other trafficking pathways involving the TGN or endosomes, indicating specificity. Compound treatment also altered localization of AP-1 in mammalian cells. These previously uncharacterized inhibitors will be useful for future studies of clathrin-mediated transport in yeast, and potentially in other organisms. Furthermore, the easily automated technology should be adaptable for identification of inhibitors of other cellular processes.
小分子抑制剂为表征高度动态和复杂的真核细胞通路（如介导膜运输的通路）提供了强大工具。然而，尽管有各种各样的化学文库，但缺乏简便且通用的检测方法限制了新型抑制剂的鉴定。在此，我们报告了一种在酵母中基于生长的简便筛选策略，用于筛选通路特异性抑制剂。该方法利用特征明确的合成基因生长缺陷来指导对所选通路抑制作用敏感的细胞的遗传设计。通过这种策略，我们鉴定出一类哌嗪基苯乙酮化合物作为反式高尔基体网络（TGN）和内体之间运输的抑制剂，这种运输依赖于网格蛋白衔接复合物AP-1。这些化合物不会显著改变涉及TGN或内体的其他运输通路，表明具有特异性。化合物处理还改变了哺乳动物细胞中AP-1的定位。这些以前未被表征的抑制剂将有助于未来对酵母中网格蛋白介导的运输以及可能在其他生物体中的研究。此外，这种易于自动化的技术应该适用于鉴定其他细胞过程的抑制剂。
