Seidl Claudia, de Lima Juliana Maria, Leme Gabriel Mazzi, Pires Ananda Ferreira, Stoll Dwight R, Cardoso Carmen Lúcia
Grupo de Cromatografia de Bioafinidade e Produtos Naturais, Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.
SEPARARE Núcleo de Pesquisa Em Cromatografia, Departamento de Química, Universidade Federal de São Carlos, São Carlos, Brazil.
Front Mol Biosci. 2022 Mar 16;9:868597. doi: 10.3389/fmolb.2022.868597. eCollection 2022.
The continuous interest in discovering new bioactive molecules derived from natural products (NP) has stimulated the development of improved screening assays to help overcome challenges in NP-based drug discovery. Here, we describe a unique platform for the online screening of acetylcholinesterase inhibitors without the need for pre-treating the sample. In the current study, we have demonstrated the ability to combine reversed-phase separation with a capillary immobilized enzyme reactor (cIMER) in two-dimensional liquid chromatography system coupled with mass spectrometry detection. We systematically investigated the effects of method parameters that are of practical significance and are known to affect the enzyme assay and interfere in the analysis such as: bioreactor dimensions, loop sizes, amount of immobilized enzyme, second dimension flow rates, reaction time, substrate concentration, presence of organic modifier, limit of detection and signal suppression. The performance of this new platform was evaluated using a mixture containing three known AChE inhibitors (tacrine, galanthamine and donepezil) and an ethanolic extract obtained from the dry bulbs of (Amaryllidaceae) was investigated to provide a proof of concept of the applicability of the platform for the analysis of complex mixtures such as those derived from NPs.
对发现源自天然产物（NP）的新生物活性分子的持续关注，刺激了改进筛选分析方法的发展，以帮助克服基于NP的药物发现中的挑战。在此，我们描述了一个独特的平台，用于在线筛选乙酰胆碱酯酶抑制剂，而无需对样品进行预处理。在当前研究中，我们展示了在二维液相色谱系统中结合反相分离与毛细管固定化酶反应器（cIMER）并联用质谱检测的能力。我们系统地研究了具有实际意义且已知会影响酶分析并干扰分析的方法参数的影响，例如：生物反应器尺寸、定量环大小、固定化酶量、第二维流速、反应时间、底物浓度、有机改性剂的存在、检测限和信号抑制。使用含有三种已知乙酰胆碱酯酶抑制剂（他克林、加兰他敏和多奈哌齐）的混合物评估了这个新平台的性能，并研究了从（石蒜科）干燥鳞茎中获得的乙醇提取物，以证明该平台适用于分析复杂混合物（如源自天然产物的混合物）的概念。
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