Mass spectrometry-based biochemical assays for enzymeinhibitor screening
Mass spectrometry-based biochemical assays for enzymeinhibitor screening
复制标题
DOI：
10.1016/j.trac.2007.08.004
复制
发表时间：
2007-10-01
影响因子：
13.1
通讯作者：
Irth, Hubertus
中科院分区：
文献类型：
--
作者：
de Boer, Arjen R.;Lingeman, Henk;Irth, Hubertus
筛选药理学相关酶的抑制剂在许多情况下是药物发现的重要起点。虽然基于荧光的检测技术在高通量筛选中发挥着重要作用，但近年来基于质谱（MS）的测定也变得越来越重要。本文综述了基于MS的酶抑制剂筛选方法的不同形式和应用，其中基于MS的酶抑制剂筛选方法可分为直接筛选和间接筛选。直接筛选方法分离并检测酶-抑制剂复合物或抑制剂，以证明对药物靶标的活性。形成酶-抑制剂复合物的活性化合物可以作为完整复合物（直接输注）通过MS直接分析，或者在通过毛细管等电聚焦（CIEF）、前沿亲和色谱（FAC）、尺寸排阻色谱（SEC）、固定化酶或超滤（UF）和解离与未结合（非活性）化合物分离后分析。间接筛选方法依赖于检测报告分子来测量分析物的酶抑制活性。当使用天然底物作为报告分子时，该测定提供功能性反应，而不是仅依赖于抑制剂结合的检测。活性的间接筛选通过与MS偶联的连续流系统（CFS）、流动注射分析（FIA）、高效液相色谱（HPLC）或基质辅助激光解吸/电离（MALDI）进行。基于MS的筛选方法可以筛选各种样品（例如，组合文库和天然产物提取物），筛选大型文库，提供活性信息、质量信息、结构信息和亲和性数据，并实现高通量。(c)2007爱思唯尔有限公司保留所有权利。
Screening for inhibitors of pharmacologically-relevant enzymes is in many cases an important starting point in drug discovery. While fluorescence-based detection techniques play an important role in high-throughput screening, mass spectrometry (MS)-based assays have gained in importance in recent years. The current review focuses on the different assay formats and applications of MS-based enzyme-inhibitor screening.MS-based assays can be categorized into direct and indirect screening methods. Direct screening methods isolate and detect the enzyme-inhibitor complex or the inhibitor to prove activity against the drug target. Active compounds that form enzyme-inhibitor complexes can be analyzed directly by MS as intact complex (direct infusion), or after isolation from the unbound (nonactive) compounds by capillary isoelectric focusing (CIEF), frontal affinity chromatography (FAC), size-exclusion chromatography (SEC), immobilized enzymes or ultrafiltration (UF), and dissociation. Indirect screening methods rely on detection of reporter molecules to measure enzyme-inhibitory activity of analytes. When using native substrates as reporter molecules, the assay provides a functional response rather than relying on detection of inhibitor binding alone. Indirect screening for activity is performed by continuous-flow systems (CFSs), flow-injection analysis (FIA), high-performance liquid chromatography (HPLC) or matrix-assisted laser desorption/ionization (MALDI), coupled to MS.MS-based screening methods can screen various kinds of samples (e.g., combinatorial libraries and natural-product extracts), screen large libraries, provide activity information, mass information, structural information and affinity data, and achieve high throughputs. (c) 2007 Elsevier Ltd. All rights reserved.