Hu Xiaolan, Wu Jian-Lin, He Quan, Xiong Zhi-Qi, Li Na
State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau SAR, 999078, China.
College of Environment and Climate, Institute of Mass Spectrometry and Atmospheric Environment, Guangdong Provincial Key Laboratory of Speed Capability Research, Jinan University, Guangzhou, 510632, China.
J Pharm Anal. 2025 Mar;15(3):101045. doi: 10.1016/j.jpha.2024.101045. Epub 2024 Jul 18.
Targeted covalent inhibitors, primarily targeting cysteine residues, have attracted great attention as potential drug candidates due to good potency and prolonged duration of action. However, their discovery is challenging. In this research, a database-assisted liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy was developed to quickly discover potential cysteine-targeting compounds. First, compounds with potential reactive groups were selected and incubated with -acetyl-cysteine in microsomes. And the precursor ions of possible cysteine-adducts were predicted based on covalent binding mechanisms to establish in-house database. Second, substrate-independent product ions produced from -acetyl-cysteine moiety were selected. Third, multiple reaction monitoring scan was conducted to achieve sensitive screening for cysteine-targeting compounds. This strategy showed broad applicability, and covalent compounds with diverse structures were screened out, offering structural resources for covalent inhibitors development. Moreover, the screened compounds, norketamine and hydroxynorketamine, could modify synaptic transmission-related proteins , indicating their potential as covalent inhibitors. This experimental-based screening strategy provides a quick and reliable guidance for the design and discovery of covalent inhibitors.
靶向共价抑制剂主要靶向半胱氨酸残基，因其具有良好的效力和延长的作用持续时间，作为潜在的药物候选物备受关注。然而，它们的发现具有挑战性。在本研究中，开发了一种数据库辅助的液相色谱 - 串联质谱（LC-MS/MS）策略，以快速发现潜在的靶向半胱氨酸的化合物。首先，选择具有潜在反应性基团的化合物，并在微粒体中与N - 乙酰半胱氨酸孵育。然后根据共价结合机制预测可能的半胱氨酸加合物的前体离子，以建立内部数据库。其次，选择由N - 乙酰半胱氨酸部分产生的与底物无关的产物离子。第三，进行多反应监测扫描，以实现对靶向半胱氨酸化合物的灵敏筛选。该策略显示出广泛的适用性，筛选出了具有不同结构的共价化合物，为共价抑制剂的开发提供了结构资源。此外，筛选出的化合物去甲氯胺酮和羟基去甲氯胺酮可以修饰与突触传递相关的蛋白质，表明它们作为共价抑制剂的潜力。这种基于实验的筛选策略为共价抑制剂的设计和发现提供了快速可靠的指导。
