使用 ITCcalc 为药物化学家提供先进的等温滴定量热法,Journal of Chemical Education

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使用 ITCcalc 为药物化学家提供先进的等温滴定量热法
Journal of Chemical Education ( IF 2.5 ) Pub Date : 2024-02-08 , DOI: 10.1021/acs.jchemed.3c01133
Stefan J. Hammerschmidt ₁ , Fabian Barthels ₁ , Annabelle C. Weldert ₁ , Christian Kersten _{1,

2}

Affiliation

等温滴定量热法 (ITC) 是热力学结合分析的首选方法。作为一种无标记溶液内技术，ITC 实验不需要修饰或固定的蛋白质或配体，因此可以在接近天然的环境条件下进行。它直接跟踪分子相互作用释放的热量，不需要竞争的报告分子。然而，经常需要先进的实验 ITC 设置来导出药物化学应用的热力学参数。除了 1:1 蛋白质-配体结合之外，复杂系统还包括置换滴定，以确定亲和力过高或过低而无法从直接滴定中得出的配体的热力学、结合时质子化变化的表征或热容量的阐明。我们为研究生药物化学家和熟悉标准 ITC 方法的学生提供实践实验，可靠地强调这些问题及其各自的解决方案。该装置可以是生化胰蛋白酶配体系统，也可以是更具成本效益的基于 EDTA 的变体。最佳实践协议的演示与可免费访问的 ITC 网络计算器ITCcalc (https://itccalc.uni-mainz.de) 结合在一起，指导对所获得的实验结果进行基础评估。

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Advanced Isothermal Titration Calorimetry for Medicinal Chemists with ITCcalc

Isothermal titration calorimetry (ITC) is the method of choice for thermodynamic binding profiling. As a label-free in-solution technique, ITC experiments do not require modified or immobilized proteins or ligands and hence can be performed under close-to-native environmental conditions. It directly follows the released heat of the molecular interaction and does not need competing reporter molecules. However, advanced experimental ITC setups are frequently necessary to derive thermodynamic parameters for medicinal chemistry applications. Beyond 1:1 protein–ligand binding, complex systems include displacement titrations to determine thermodynamics for ligands with affinities either too high or too low to be derived from direct titrations, the characterization of protonation changes upon binding, or the elucidation of heat capacities. We present hands-on experiments for graduate medicinal chemists and students familiar with standard ITC methods, reliably highlighting these issues and their respective solutions. The setup can be either a biochemical trypsin–ligand system or a more cost-efficient EDTA-based variant. This demonstration of best practice protocols comes together with the freely accessible ITC web calculator ITCcalc (https://itccalc.uni-mainz.de), guiding through the underlying evaluation of the obtained experimental results.

更新日期：2024-02-08

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