Dirnberger Dietmar, Unsin Gabriele, Schlenker Stephan, Reichel Christoph
GPC Biotech AG, Fraunhoferstrasse 20, 82152 Martinsried, Germany.
Chembiochem. 2006 Jun;7(6):936-42. doi: 10.1002/cbic.200500544.
The identification of receptors for small molecules is of great pharmaceutical importance for drug-discovery research. Several systems for the identification of protein-small-molecule interactions have been developed in the past. These were modifications of the classical yeast two-hybrid system, relying on a transcriptional read-out following nuclear translocation of the complex. Here we present a novel three-hybrid technology based on the split-ubiquitin system for the analysis of protein-small-molecule interactions independently of a nuclear translocation of the complex. The performance of the system is compared to a method based on the classical yeast two-hybrid system by using a chemical inducer of dimerization (CID) comprised of methotrexate linked to dexamethasone. Steric issues are addressed by varying the linker length of the compounds, as well as by comparing the orientation of fusion proteins. The system is further extended to the analysis of a small-molecule inhibitor of human PCTAIRE protein kinase 3, which is related to cyclin-dependent kinases (CDKs), an important class of pharmaceutical targets.
小分子受体的鉴定对于药物研发研究具有重大的药学意义。过去已开发出多种用于鉴定蛋白质 - 小分子相互作用的系统。这些系统是经典酵母双杂交系统的改良版本，依赖于复合物核转位后的转录读数。在此，我们提出一种基于分裂泛素系统的新型三杂交技术，用于独立于复合物核转位来分析蛋白质 - 小分子相互作用。通过使用由与地塞米松连接的甲氨蝶呤组成的二聚化化学诱导剂（CID），将该系统的性能与基于经典酵母双杂交系统的方法进行比较。通过改变化合物的连接子长度以及比较融合蛋白的方向来解决空间问题。该系统进一步扩展至分析与细胞周期蛋白依赖性激酶（CDK，一类重要的药物靶点）相关的人类PCTAIRE蛋白激酶3的小分子抑制剂。
