Kranz James K, Schalk-Hihi Celine
Biopharmaceutical Technologies, GlaxoSmithKline Biopharmaceutical Research and Development, Upper Merion, Pennsylvania, USA.
Methods Enzymol. 2011;493:277-98. doi: 10.1016/B978-0-12-381274-2.00011-X.
Measuring the strength of binding of low molecular weight ligands to a target protein is a significant challenge to fragment-based drug discovery that must be solved. Thermal shift assays are uniquely suited for this purpose, due to the thermodynamic effects of a ligand on protein thermal stability. We show here how to implement a thermal shift assay, describing the basic features and analysis of the protein unfolding data. We then describe in detail the effects of a ligand on the observed stability of the protein to produce a shift in stability. The anatomy of ligand-induced thermal shift data is discussed in detail. We describe the unique aspects of concentration-response curves, the effect of protein unfolding energetics, and the stoichiometry of the interaction. We outline a typical assay development strategy for optimizing dye type and concentration, protein concentration, and buffer conditions. Guidelines are presented to demonstrate the limits of detection for weak-binding ligands, as applied to sulfonamide-based inhibitors of carbonic anhydrase II and applied to nucleotide binding to the death-associated protein kinase 1 catalytic domain.
测量低分子量配体与靶蛋白的结合强度是基于片段的药物发现中一项必须解决的重大挑战。由于配体对蛋白质热稳定性的热力学效应，热位移分析特别适合于此目的。我们在此展示如何实施热位移分析，描述蛋白质解折叠数据的基本特征和分析方法。然后，我们详细描述配体对观察到的蛋白质稳定性的影响，以产生稳定性的变化。详细讨论了配体诱导的热位移数据的剖析。我们描述了浓度-响应曲线的独特方面、蛋白质解折叠能量学的影响以及相互作用的化学计量。我们概述了一种典型的分析方法开发策略，用于优化染料类型和浓度、蛋白质浓度以及缓冲条件。给出了指导方针，以证明对弱结合配体的检测限，应用于碳酸酐酶II的磺酰胺类抑制剂以及应用于核苷酸与死亡相关蛋白激酶1催化结构域的结合。
