Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells.
Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells.
复制标题
DOI：
10.3389/fendo.2012.00100
复制
发表时间：
2012
影响因子：
5.2
通讯作者：
Deprez B
中科院分区：
文献类型：
--
作者：
Couturier C;Deprez B
细胞生命的每一步及其对环境的反应或适应都是由称为“相互作用组”的蛋白质/蛋白质相互作用网络介导的。由于专门研究这些相互作用的敏感技术的发展，我们对这个网络的了解不断增长。生物发光共振能量转移（BRET）技术主要用于动态监测活细胞中的蛋白质/蛋白质相互作用（PPI），并已广泛用于研究哺乳动物细胞中受体和活化复合物分子内或分子外构象变化引起的受体活化。一些相互作用被描述为在人类病理过程中至关重要，最近出现了一类针对它们的新药物。BRET方法非常适合于识别PPI的抑制剂，这里描述了为什么以及如何建立和优化基于BRET的高通量筛选试验来寻找这样的抑制性化合物。审查在哺乳动物细胞中开发此类BRET测定时要考虑的不同参数，以提供一般指南：考虑靶向相互作用、BRET版本的选择、相互作用的诱导、监测相互作用的动力学和BRET阅读、底物浓度、细胞数量和培养基组成对Z '因子的影响，以及来自有色或荧光化合物的预期干扰。
Each step of the cell life and its response or adaptation to its environment are mediated by a network of protein/protein interactions termed “interactome.” Our knowledge of this network keeps growing due to the development of sensitive techniques devoted to study these interactions. The bioluminescence resonance energy transfer (BRET) technique was primarily developed to allow the dynamic monitoring of protein/protein interactions (PPI) in living cells, and has widely been used to study receptor activation by intra- or extra-molecular conformational changes within receptors and activated complexes in mammal cells. Some interactions are described as crucial in human pathological processes, and a new class of drugs targeting them has recently emerged. The BRET method is well suited to identify inhibitors of PPI and here is described why and how to set up and optimize a high throughput screening assay based on BRET to search for such inhibitory compounds. The different parameters to take into account when developing such BRET assays in mammal cells are reviewed to give general guidelines: considerations on the targeted interaction, choice of BRET version, inducibility of the interaction, kinetic of the monitored interaction, and of the BRET reading, influence of substrate concentration, number of cells and medium composition used on the Z′ factor, and expected interferences from colored or fluorescent compounds.