A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein - Protein interactions by fluorescence polarization
A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein - Protein interactions by fluorescence polarization
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DOI：
10.1021/bi048233g
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发表时间：
2004-12-28
期刊：
影响因子：
2.9
通讯作者：
Wagner, G
中科院分区：
文献类型：
--
作者：
Roehrl, MHA;Wang, JY;Wagner, G
平衡结合实验被广泛用于精确表征生物系统中的结合和竞争结合行为。现代化学生物学中的高通量发现工作在很大程度上依赖于这一原则。在这里，我们推导出精确的解析表达式一般的竞争性结合模型，也可以解释一个常见的现象，在这些类型的实验，反合作不完全位移。我们探讨了非特异性结合行为和参数错误估计的影响。所有的表达式都是根据先验确定的总浓度导出的。我们讨论了基于荧光偏振的高通量筛选试验的一般框架和试验开发，灵敏度制度，数据质量控制，分析和排名的策略。理论研究结果可视化模拟使用现实的参数集。我们的研究结果是发现人钙调磷酸酶和NFAT转录因子之间蛋白质-蛋白质相互作用的小分子抑制剂的基础，如随后的论文中所讨论的（31）。
Equilibrium binding experiments are widely used for the accurate characterization of binding and competitive binding behavior in biological systems. Modern high-throughput discovery efforts in chemical biology rely heavily upon this principle. Here, we derive exact analytical expressions for general competitive binding models which can also explain a commonly encountered phenomenon in these types of experiments, anticooperative incomplete displacement. We explore the effects of nonspecific binding behavior and parameter misestimation. All expressions are derived in terms of total concentrations determined a priori. We discuss a general framework for high-throughput screening assays based on fluorescence polarization and strategies for assay development, sensitivity regimes, data quality control, analysis, and ranking. Theoretical findings are visualized by simulations using realistic parameter sets. Our results are the basis for the discovery of small-molecule inhibitors of the protein-protein interaction between human calcineurin and NFAT transcription factors, as discussed in the subsequent paper (31).