Alzheimer’s disease is the most common cause of dementia, afflicting over 34 million patients worldwide. Since β-secretase is a rate-limiting enzyme of the production of neurotoxic β-amyloid peptide oligomers abnormally accumulated in the affected brain tissue, its specific inhibition appears to be a promising approach to slowing down or even stopping the progression of the disease. Hence two on-line capillary electrophoretic methods for studies of β-secretase activity based on the principles of transverse diffusion of laminar flow profiles and electrophoretically mediated microanalysis were developed, both using a simple unlabeled peptide substrate and UV detection. The optimized procedures were thoroughly validated and applied for determining the enzyme’s kinetic parameters and the inhibition characteristics of two potent probe inhibitors. The resulting values were found to be comparable to literature data obtained with other analytical techniques. The suitability of the employed methodologies for different experimental designs is discussed on the basis of a statistical evaluation of the experimental data. The presented methods constitute a miniaturized and fully automated tool, which should be suitable for kinetic and inhibition studies of β-secretase as a target for Alzheimer’s disease drug discovery in the early stages of the development of a new drug.

阿尔茨海默氏病是痴呆症的最常见原因，全世界折磨了3400万患者。由于β-分泌酶是在受影响的脑组织中异常积累的神经毒性β-淀粉样肽寡聚体产生的限速酶，因此其特异性抑制作用似乎是减缓甚至阻止疾病进展的一种有前途的方法。因此，基于简单的未标记肽底物和紫外检测技术，开发了两种基于层流剖面横向扩散原理和电泳介导的微量分析的研究β-分泌酶活性的在线毛细管电泳方法。对优化的程序进行了充分验证，并将其用于确定酶的动力学参数和两种有效探针抑制剂的抑制特性。发现结果值可与使用其他分析技术获得的文献数据相媲美。在对实验数据进行统计评估的基础上，讨论了所采用方法对不同实验设计的适用性。提出的方法构成了一种小型且全自动的工具，该工具应适合于在新药开发的早期阶段以β-分泌酶作为阿尔茨海默氏病药物发现目标的动力学和抑制研究。