Vilela Adriana Ferreira Lopes, Seidl Cláudia, Lima Juliana Maria, Cardoso Carmen Lúcia
Departamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil.
Departamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil.
Anal Biochem. 2018 May 15;549:53-57. doi: 10.1016/j.ab.2018.03.012. Epub 2018 Mar 14.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H]m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC values were 0.03 ± 0.006 μM and 0.88 ± 0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ± 0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results.
乙酰胆碱酯酶（AChE）和丁酰胆碱酯酶（BChE）是关键的胆碱酯酶，负责将乙酰胆碱水解为胆碱和乙酸，这是胆碱能神经元恢复的重要过程。中枢神经系统中胆碱能功能的丧失会导致与衰老和阿尔茨海默病（AD）相关的认知衰退。抑制试验在药物发现过程中起着重要作用。在此，我们描述了一种改进的无标记方法，用于筛选和表征新的BChE配体。液相色谱系统使用固定化毛细管酶反应器（ICER）作为与质谱仪（MS）联用的低亲和力和高选择性柱。通过在短时间内监测胆碱的前体离子[M + H]m/z 104来评估酶活性。该方法使用两种已知的胆碱酯酶抑制剂他克林和加兰他敏进行了验证。他克林和加兰他敏的IC值分别为0.03±0.006μM和0.88±0.2，加兰他敏的Ki为0.11±0.2。huBChE-ICER与灵敏的酶分析检测（如MS）的有效结合，提高了新配体可靠、快速的鉴定。此外，产物的特异性直接定量有助于减少假阳性和假阴性结果。
