Target identification by small-molecule labeling in live cells

描述（由申请人提供）：该项目的长期目标是开发一种强大的蛋白质组学方法，以确定活细胞中小分子化合物和药物的靶点。表型筛选通常用于鉴定新型生物活性小分子探针和开发新的治疗方法。正向化学基因组学筛选的主要瓶颈是新型小分子探针的靶点识别和验证。引人注目的是， 对于许多FDA批准的药物和小分子“工具”化合物来说，仍然是未知的。识别治疗靶点和“脱靶”允许优化化合物的选择性，并应减少最终药物的潜在副作用。我们的建议描述了一种新的策略，使用小分子抑制剂作为体内靶向部分，生物素化其分子靶标及其蛋白质复合物，用于蛋白质组学鉴定。这种新方法与传统的生物化学方法不同，可以识别亲和力弱得多的结合剂和更高阶的蛋白质-蛋白质相互作用。由于我们的方法探索了当小分子探针与其蛋白质靶点结合时多蛋白质复合物中的空间关系，因此可以提供一种新的视角来表征小分子的作用机制。

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a robust proteomics approach to identify targets of small-molecule compounds and drugs in live cells. Phenotypic screens are commonly used to identify novel bioactive small-molecule probes and develop new therapeutic approaches. The major bottleneck in forward chemical-genomics screens is target identification and validation of novel small-molecule probes. Strikingly, targets are still unknown for many FDA-approved drugs and small-molecule "tool" compounds. Identifying both the therapeutic targets and "off targets" allows optimization of a compound's selectivity and should decrease potential side effects in the eventual drug. Our proposal describes a novel strategy to use small-molecule inhibitors as in vivo targeting moieties to biotinylate their molecular targets and their protein complexes for proteomic identification. This novel approach is distinct from traditional biochemical approaches and may allow the identification of much weaker affinity binders and higher order protein-protein interactions. As our approach explores spatial relationships in multi-protein complexes when a small-molecule probe is bound to its protein target, it may provide a novel perspective to characterize the small molecule's mechanisms of action.