Zheng Pingyi, Wang Xinru, Tang Ruijun, Cui Yuchen, Yuan Xiaoqian, Li Youxin
Tianjin Key Laboratory for Modern Drug Delivery and High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Faculty of Medicine, Tianjin University, China.
Tianjin Key Laboratory for Modern Drug Delivery and High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Faculty of Medicine, Tianjin University, China; Neurocritical Care Medicine Innovation Center, Ministry of Education, Tianjin University, China; State Key Laboratory of Advanced Medical Materials and Devices, Tianjin University, China.
Int J Biol Macromol. 2025 Sep;321(Pt 4):146545. doi: 10.1016/j.ijbiomac.2025.146545. Epub 2025 Aug 4.
Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) are of great significance in the study of the fine regulation of purine metabolism. It will provide an important basis for the study of the pathogenesis of immunodeficiency diseases, leukemia and other diseases related to purine metabolism abnormality, as well as for the target of drug discovery and development. Here, a new online capillary electrophoresis and post-calculation strategy was proposed for the quick determination of adenosine deaminase and purine nucleoside phosphorylase kinetic constant and their inhibitors screening. With unique injection procedure, special separation method and innovative calculation strategy, the enzyme kinetics could be obtained through combing the product curves containing enzyme reaction kinetics in a CE run. Furthermore, the weak product signal and the difficult separation issues for inosine, adenosine and hypoxanthine under the neutral online enzyme reaction conditions were effectively solved for the first time by the adjusted gravity mediated-capillary electrophoresis (AGM-CE). Compared with the traditional offline method, the proposed strategy was reliable and efficient. It also was successfully used in inhibitor screening of adenosine deaminase and purine nucleoside phosphorylase. Results proved that the new strategy had great potential in enzyme analysis and inhibitor screening.
腺苷脱氨酶（ADA）和嘌呤核苷磷酸化酶（PNP）在嘌呤代谢精细调节的研究中具有重要意义。它将为免疫缺陷疾病、白血病及其他与嘌呤代谢异常相关疾病的发病机制研究，以及药物研发靶点提供重要依据。在此，提出了一种新的在线毛细管电泳及计算后策略，用于快速测定腺苷脱氨酶和嘌呤核苷磷酸化酶的动力学常数及其抑制剂筛选。通过独特的进样程序、特殊的分离方法和创新的计算策略，在一次毛细管电泳运行中结合包含酶反应动力学的产物曲线即可获得酶动力学。此外，通过调整重力介导毛细管电泳（AGM-CE）首次有效解决了中性在线酶反应条件下肌苷、腺苷和次黄嘌呤产物信号弱及分离困难的问题。与传统离线方法相比，该策略可靠且高效。它还成功用于腺苷脱氨酶和嘌呤核苷磷酸化酶的抑制剂筛选。结果证明，该新策略在酶分析和抑制剂筛选方面具有巨大潜力。
