Department of Chemistry , McGill University , H3A 0B8 Montreal , Canada.
Anal Chem. 2018 Jul 17;90(14):8430-8435. doi: 10.1021/acs.analchem.8b00993. Epub 2018 Jul 6.
Techniques for rapidly measuring both the strength and mode of enzyme inhibitors are crucial to lead generation and optimization in drug development. Isothermal titration calorimetry (ITC) is emerging as a powerful tool for measuring enzyme kinetics with distinct advantages over traditional techniques. ITC measures heat flow, a feature of nearly all chemical reactions, and gives an instantaneous readout of enzyme velocity, eliminating the need for artificial substrates or postreaction processing. In principle, ITC is an ideal method for characterizing enzyme inhibition. However, existing ITC experiments are not well-suited to rapid throughput and few studies to date have employed this approach. We have developed a new ITC experiment, in which substrate and inhibitor are premixed in the injection syringe, that yields complete kinetic characterization of an enzyme inhibitor in an hour or less. This corresponds to savings in time and material of 5-fold or greater compared to previous ITC methods. We validated the approach using the trypsin inhibitor benzamidine as a model system, recapitulating both its competitive inhibition mode and binding constant. Our approach combines the rapid throughput of optimized spectroscopic assays with the universality and precision of ITC-based methods, providing substantially improved inhibitor characterization for biochemistry and drug development applications.
快速测量酶抑制剂的强度和模式的技术对于药物开发中的先导化合物生成和优化至关重要。等温滴定量热法（ITC）作为一种强大的测量酶动力学的工具正在兴起，与传统技术相比具有明显的优势。ITC 测量热流量，这是几乎所有化学反应的一个特征，并提供酶速度的即时读数，消除了对人工底物或反应后处理的需求。原则上，ITC 是一种理想的酶抑制特征描述方法。然而，现有的 ITC 实验并不适合快速高通量，迄今为止很少有研究采用这种方法。我们开发了一种新的 ITC 实验，其中底物和抑制剂在注射针筒中预先混合，在一小时或更短的时间内即可完成对酶抑制剂的完整动力学特征描述。与以前的 ITC 方法相比，这在时间和材料上节省了 5 倍或更多。我们使用胰蛋白酶抑制剂苯甲脒作为模型系统验证了该方法，重现了其竞争性抑制模式和结合常数。我们的方法将优化的光谱测定法的高通量与基于 ITC 的方法的普遍性和精确性结合在一起，为生物化学和药物开发应用提供了大大改进的抑制剂特征描述。
