The Anhui Provincial Key Laboratory of High Field Magnetic Resonance Image, High Magnetic Field Laboratory, Chinese Academy of Science, Hefei, Anhui 230031, P. R. China.
The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Center for BioAnalytical Chemistry, and Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China.
Chem Commun (Camb). 2021 Sep 21;57(75):9602-9605. doi: 10.1039/d1cc03301h.
Herein, we report an EPR-based method for protease enzymatic characterization and inhibitor screening. This method utilizes dual paramagnetically-labeled probes consisting of a nitroxide spin probe and a Gd ion flanking a peptide that could be specifically cleaved by protease caspase-3. Distance-dependent dipolar coupling between the two paramagnetic centers can be modulated by the protease cleavage activity, thus providing a straightforward and convenient method for protease activity detection using EPR spectroscopy under ambient conditions. Moreover, time-course monitoring of the protease-catalyzed cleavage reaction demonstrated that this EPR-based method could not only allow a direct quantitative enzymatic kinetic assessment, but also could be used for protease inhibitor screening, thus holding great potential in drug discovery studies.
在此，我们报告了一种基于电子顺磁共振（EPR）的蛋白酶酶学特征分析和抑制剂筛选方法。该方法利用双顺磁标记探针，由一个氮氧自由基探针和一个镓离子组成，两侧连接一段可被蛋白酶 caspase-3 特异性切割的肽段。两个顺磁中心之间的距离相关偶极耦合可被蛋白酶的切割活性调节，因此提供了一种在环境条件下使用 EPR 光谱进行蛋白酶活性检测的简单方便的方法。此外，对蛋白酶催化切割反应的时程监测表明，这种基于 EPR 的方法不仅可以进行直接的定量酶动力学评估，还可以用于蛋白酶抑制剂筛选，因此在药物发现研究中具有很大的潜力。
