Kool Jeroen, van Liempd Sebastiaan M, Ramautar Rawi, Schenk Tim, Meerman John H N, Irth Hubertus, Commandeur Jan N M, Vermeulen Nico P E
LACDR-Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, the Netherlands.
J Biomol Screen. 2005 Aug;10(5):427-36. doi: 10.1177/1087057105274904.
A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the online Cyt P450 enzyme affinity detection (EAD) system was optimized for enzyme (beta-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 (alpha-naphthoflavone, beta-naphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC50 values were online in FIA-mode determined and compared with those obtained with standardmicrosomal assay conditions. The IC50 values obtained with the online Cyt P450 EAD system agreed well with the IC50 values obtained in the standard assays. For high affinity ligands of Cyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n=3; signal to noise [S/N]=3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n=1; S/N=3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.
本文描述了一种高分辨率筛选平台，该平台将哺乳动物细胞色素P450（Cyt P450）的在线亲和检测与梯度反相高效液相色谱（HPLC）相结合。为此，针对酶（β-萘黄酮诱导的大鼠肝微粒体）、探针底物（乙氧萘胺）和有机改性剂（甲醇或乙腈）对在线Cyt P450酶亲和检测（EAD）系统进行了优化。优化后的Cyt P450 EAD系统首先在流动注射分析（FIA）模式下，用7种已知的Cyt P450 1A1/1A2配体（α-萘黄酮、β-萘黄酮、椭圆玫瑰树碱、9-羟基椭圆玫瑰树碱、氟伏沙明、咖啡因和非那西丁）进行了评估。随后，在FIA模式下在线测定IC50值，并与标准微粒体测定条件下获得的值进行比较。在线Cyt P450 EAD系统获得的IC50值与标准测定中获得的IC50值吻合良好。对于Cyt P450 1A1/1A2的高亲和力配体，进样检测限为1至3 pmol（n = 3；信噪比[S/N]=3）。随后，使用与梯度HPLC在线联用的优化Cyt P450 EAD系统，研究了配体混合物中各配体的抑制特性。使用集成的在线梯度HPLC Cyt P450 EAD平台，高亲和力配体的进样检测限为10至25 pmol（n = 1；S/N = 3）。结论是，这种新型筛选技术为快速、灵敏地筛选对肝微粒体Cyt P450具有亲和力的混合物中的单个化合物提供了新的视角。
