Redhead Martin, Satchell Rupert, McCarthy Ciara, Pollack Scott, Unitt John
Bioscience Department, Sygnature Discovery , Nottingham NG1 1GF, U.K.
Biochemistry. 2017 Nov 28;56(47):6187-6199. doi: 10.1021/acs.biochem.7b00860. Epub 2017 Nov 15.
Thermal shift assays (TSAs) are among the most commonly used biophysical approaches in drug discovery in both academic and industrial settings. However, the most common interpretation of the data generated by a TSA is purely qualitative, using only the change in melting temperature (ΔT) as a metric. This has left many questions surrounding the interpretation of the data as well as whether the TSA truly correlates with other assays. TSAs also lack theoretical descriptions of the melt behavior of proteins in the presence of multiple ligands. Here we describe a novel simplified analytical framework based on "pseudoisothermal" models as well as exact thermodynamic descriptions of protein-ligand melt behavior rooted in changes in the entropy of melting. We show how the models are broad and independently applicable, in that they can describe the behavior of any macromolecule such as a protein or DNA and demonstrate good correlations with other techniques. These models are shown to give good descriptions of assay systems containing single or multiple ligands and can determine the mechanism of interaction. The models are derived from first principles, and the theoretical justification is discussed.
热位移分析（TSA）是学术和工业环境中药物发现中最常用的生物物理方法之一。然而，对TSA产生的数据最常见的解释纯粹是定性的，仅将解链温度的变化（ΔT）用作衡量标准。这使得围绕数据解释以及TSA是否真的与其他分析方法相关存在许多问题。TSA也缺乏对存在多种配体时蛋白质熔解行为的理论描述。在此，我们描述了一种基于“伪等温”模型的新型简化分析框架，以及基于熔解熵变化的蛋白质 - 配体熔解行为的精确热力学描述。我们展示了这些模型具有广泛的适用性且可独立应用，因为它们可以描述任何大分子（如蛋白质或DNA）的行为，并与其他技术表现出良好的相关性。这些模型被证明能够很好地描述包含单个或多个配体的分析系统，并能确定相互作用的机制。这些模型是从第一原理推导出来的，并对其理论依据进行了讨论。
