Schobel U, Frenay M, van Elswijk D A, McAndrews J M, Long K R, Olson L M, Bobzin S C, Irth H
ScreenTec B.V., Leiden, The Netherlands.
J Biomol Screen. 2001 Oct;6(5):291-303. doi: 10.1177/108705710100600503.
A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) alpha and beta binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flowthrough fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.
本文描述了一种新的筛选技术，该技术将生化分析与高效液相色谱（HPLC）的分离能力相结合，在此称为高分辨率筛选（HRS）技术。通过筛选植物天然产物提取物库中的雌激素受体（ER）α和β结合活性，证明了HRS技术分析复杂混合物中生物活性化合物的能力。通过将HRS系统与质谱（MS）联用，实现了粗提物中生物活性成分的同时结构解析。与传统的微量滴定型生物测定法不同，提取物与ER以及所用标记物香豆雌酚的相互作用在一个封闭的连续流动反应检测系统中快速进行，该系统直接连接到HPLC分离柱的出口。使用流通式荧光检测器在线检测这种均相荧光增强型测定法的反应产物。提取物库的初步筛选在快速流动注射分析模式（FlowScreening）下进行，其中色谱分离系统被旁路。使用两条检测线并行，对文库进行了高速筛选。在基于传统96孔微孔板的荧光偏振测定法中，总共98%的鉴定命中物得到了确认，这表明了FlowScreening过程的可靠性。对活性提取物进行转录激活测定，以评估生物活性提取物的功能活性。只有功能活性提取物在更耗时的与MS联用的HRS模式下进行处理。从50%的功能活性提取物中实时获得了有关活性化合物数量、保留时间、分子量以及作为其生物活性函数的MS/MS指纹的信息。与传统的分级分离方法相比，这大大提高了天然产物提取物中生物活性化合物表征的速度。
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