Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging.
Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging.
复制标题
DOI：
10.1007/978-1-62703-311-4_11
复制
发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Vereb, Gyorgy
中科院分区：
文献类型：
--
作者：
Roszik, Janos;Toth, Gabor;Vereb, Gyorgy
蛋白质是药物发现的主要靶点，许多新药都是通过破坏蛋白质-蛋白质相互作用来发挥作用的。通常通过生物化学方法验证分子相互作用的抑制，然而，当相互作用不够稳定时，这些方法通常不可行。荧光共振能量转移（FRET）是一个很好的工具，用于确定直接分子之间的相互作用，在细胞膜或细胞内的自然状态。虽然最初建立作为流式细胞术的方法，FRET已被改编为显微镜，允许在单细胞水平的亚细胞共定位的分析。在这一章中，我们提供理论介绍的现象FRET，和协议-包括标记技术，测量和评价显微镜图像-最简单的微观FRET方法，受体光漂白FRET。这种技术通常可用于研究蛋白质相互作用，只需要一个标准的共聚焦激光扫描显微镜。为了证明基于图像的FRET用于测试蛋白质-蛋白质相互作用的药理学破坏的价值，我们展示了如何使用该技术验证人源化单克隆抗体帕妥珠单抗对ErbB 2和ErbB 1的异源二聚化的抑制。
Proteins are the major targets of drug discovery and many of the new drugs are designed to exert their effect by disrupting protein-protein interactions. Validation of the inhibition of molecular interactions is generally done by biochemical methods, however, these are often not feasible when the interaction is not stable enough. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining direct molecular interactions between two molecules in the cell membrane or inside cells in their natural state. Although originally established as a flow cytometric approach, FRET has been adapted for microscopy, allowing for analysis of sub-cellular co-localization at the single cell level. In this chapter, we provide theoretical introduction to the phenomenon of FRET, and a protocol - including labeling techniques, measurement, and evaluation of microscopy images - of the simplest microscopic FRET approach, acceptor photobleaching FRET. This technique is generally usable for studying protein interactions and requires only a standard confocal laser scanning microscope. To demonstrate the value of image based FRET for testing pharmacological disruption of protein-protein interactions, we show how inhibition of the hetero-dimerization of ErbB2 and ErbB1 by the humanized monoclonal antibody pertuzumab can be validated using this technique.