Huang J, Schreiber S L
Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13396-401. doi: 10.1073/pnas.94.25.13396.
Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein-protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein-protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These "nanodroplets" contain defined media, cells, and one or more beads containing approximately 100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein-protein interactions in living cells or organisms.
细胞过程由复杂的分子相互作用网络介导。剖析它们的作用最常见的方法是使用基因突变，例如改变蛋白质 - 蛋白质相互作用。能达到相同结果的小分子将为遗传方法提供有力补充，但一般认为这类分子很罕见。然而，有几种天然产物说明了这种方法的可行性。裂分 - 混合合成现在提供了一种简单的机械手段来制备大量复杂的、甚至类似天然产物的分子，这些分子分别附着在细胞大小的聚合物珠粒上。在此，我们描述了一种与裂分 - 混合合成兼容的遗传系统，该系统允许在100至200纳升的细胞培养微滴中检测可穿透细胞的蛋白质 - 蛋白质相互作用的小分子抑制剂，这些微滴是通过一种最近描述的技术制备的，该技术可排列大量此类微滴。这些“纳米微滴”含有特定的培养基、细胞以及一个或多个珠粒，每个珠粒含有约100皮摩尔的可光释放小分子和一定数量的受控细胞。本研究中使用的工程化酿酒酵母细胞在半乳糖诱导后表达两种相互作用的蛋白质，其相互作用在5 - 氟乳清酸存在时导致细胞死亡（可诱导的反向双杂交测定）。小分子破坏这种相互作用会允许细胞生长，并且小分子可以在诱导毒性相互作用前数小时引入系统。我们证明，在纳米微滴中，纳摩尔浓度的小分子FK506可以破坏激活素受体R1和免疫亲和蛋白FKBP12之间的相互作用。该系统应提供一种通用方法，用于选择可穿透细胞的配体，可用于研究活细胞或生物体中蛋白质 - 蛋白质相互作用的相关性。
