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Development of three parallel cytochrome P450 enzyme affinity detection systems coupled on-line to gradient high-performance liquid chromatography

机译：在线与梯度高效液相色谱联用的三种平行细胞色素P450酶亲和力检测系统的开发

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摘要

A high resolution screening (HRS) technology is described, in which gradient high-performance liquid chromatography (HPLC) is connected on-line to three parallel placed bioaffinity detection systems containing mammalian cytochromes P450 (P450s). The three so-called enzyme affinity detection (EAD) systems contained, respectively, liver microsomes from rats induced by β-naphthoflavone (CYP1A activity), phenobarbital (CYP2B activity), and dexamethasone (CYP3A activity). Each P450-EAD system was optimized for enzyme, substrate, and organic modifier (isopropyl alcohol, methanol, and acetonitrile) in flow injection analysis mode. Characteristic P450 ligands were used to validate the P450-EAD systems. IC50 values of the ligands were measured and found to be similar to those obtained with conventional microtiter plate reader assays. Detection limits (n = 3; signal-to-noise ratio = 3) of potent inhibitors ranged from 1 to 3 pmol for CYP1A activity, 4 to 17 pmol for CYP2B activity, and 4 to 15 pmol for CYP3A activity. The three optimized P450-EAD systems were subsequently coupled to gradient HPLC and used to screen compound mixtures for individual ligands. Finally, to increase analysis efficiency, a HRS system was constructed in which all three P450-EAD systems were coupled on-line and in parallel to gradient HPLC. The triple parallelized P450-EAD system was shown to enable rapid profiling of individual components in complex mixtures for inhibitory activity to three different P450s. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics.
机译：描述了一种高分辨率筛选（HRS）技术，其中梯度高效液相色谱（HPLC）在线连接到三个并行放置的，包含哺乳动物细胞色素P450（P450s）的生物亲和力检测系统。这三个所谓的酶亲和力检测（EAD）系统分别包含由β-萘黄酮（CYP1A活性），苯巴比妥（CYP2B活性）和地塞米松（CYP3A活性）诱导的大鼠肝微粒体。每个P450-EAD系统都在流动注射分析模式下针对酶，底物和有机改性剂（异丙醇，甲醇和乙腈）进行了优化。使用特征性P450配体来验证P450-EAD系统。测量了配体的IC 50值，发现其与常规的微量滴定板读数器测定法相似。对于CYP1A活性，有效抑制剂的检测限（n = 3；信噪比= 3）为CYP1A活性为1至3 pmol，CYP2B活性为4至17 pmol，而CYP3A活性为4至15 pmol。随后将三个优化的P450-EAD系统与梯度HPLC耦合，并用于筛选化合物混合物中的单个配体。最后，为了提高分析效率，构建了一个HRS系统，其中所有三个P450-EAD系统都在线且与梯度HPLC并行连接。已显示三重平行化的P450-EAD系统能够快速分析复杂混合物中的各个成分，以抑制三种不同的P450。版权所有©2007，美国药理与实验治疗学会。

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