Hummel Matthew A, Tracy Timothy S, Hutzler J Matthew, Wahlstrom Jan L, Zhou Yihong, Rock Dan A
Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, USA.
J Biomol Screen. 2006 Apr;11(3):303-9. doi: 10.1177/1087057105285612. Epub 2006 Feb 20.
7-Methoxy-4-trifluoromethylcoumarin (MFC) has been used extensively in high-throughput screens for the identification of potential CYP2C9 interactions. More recently, additional probes from Invitrogen have been used. Vivid 2C9 Green is the largest of the probes and has had limited prior characterization. The new series of probes differ significantly from MFC and were examined for their ability to identify interactions with 19 CYP2C9 substrates/inhibitors. The inhibition profiles depend largely on the physical differences between the fluorescent probe substrates. Cytochrome b5 (cyt b5) was also investigated for the ability to alter the inhibition profile of a given compound. The stoichiometric addition of cyt b5 caused an increase in V max of MFC and Vivid 2C9 Green 4.4 and 1.7 times, respectively. Furthermore, cyt b5 imposes a steric component to the active site as the inhibition profiles were significantly affected in incubations with MFC. The addition of cyt b5 had limited impact on the inhibition profiles generated with Vivid 2C9 Green. The K(m) of Vivid 2C9 Green increased from 1.2+/-0.2 micro M to 4.8+/-0.3 micro Mas a result of cyt b5 addition. These results illustrate that multiple substrate probes may be necessary for screening drug-drug interaction in CYP2C9 and that cyt b5 effects can impart steric restraints on the CYP2C9 active site.
7-甲氧基-4-三氟甲基香豆素（MFC）已广泛用于高通量筛选，以鉴定潜在的CYP2C9相互作用。最近，来自英杰公司的其他探针也被使用。Vivid 2C9 Green是最大的探针，之前对其表征有限。新系列的探针与MFC有显著差异，并对它们与19种CYP2C9底物/抑制剂相互作用的识别能力进行了研究。抑制谱很大程度上取决于荧光探针底物之间的物理差异。还研究了细胞色素b5（cyt b5）改变给定化合物抑制谱的能力。化学计量添加cyt b5分别使MFC和Vivid 2C9 Green的Vmax增加了4.4倍和1.7倍。此外，由于在与MFC的孵育中抑制谱受到显著影响，cyt b5在活性位点施加了空间位阻成分。添加cyt b5对Vivid 2C9 Green产生的抑制谱影响有限。添加cyt b5后，Vivid 2C9 Green的K(m)从1.2±0.2微摩尔增加到4.8±0.3微摩尔。这些结果表明，在筛选CYP2C9中的药物-药物相互作用时可能需要多种底物探针，并且cyt b5的作用可以在CYP2C9活性位点上施加空间位阻。
