Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells
Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells
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Gomisin A 是一种新型异构体特异性探针，用于选择性传感肝微粒体和活细胞中的人细胞色素 P450 3A4
DOI：
10.1208/s12248-015-9827-4
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发表时间：
2016-01-01
期刊：
影响因子：
4.5
通讯作者：
Yang, Ling
中科院分区：
文献类型：
--
作者：
Wu, Jing-Jing;Ge, Guang-Bo;Yang, Ling
近一半的处方药由人体细胞色素P450（CYP）3A代谢。CYP3A4和CYP3A5是人体CYP3A的两种主要亚型，且底物谱大部分相同。此前一项极为有限的研究区分了CYP3A4和CYP3A5的活性，揭示了预测CYP3A介导的药物清除及药物 - 药物相互作用所面临的挑战。在本研究中，我们引入了具有二苯并环辛二烯骨架的戈米辛A（GA），将其作为CYP3A4的新型选择性探针。通过液相色谱 - 质谱联用（LC - MS）和核磁共振（NMR），GA的主要代谢产物被完全鉴定为8 - 羟基化GA。通过反应表型分析、化学抑制分析及相关性研究，确定CYP3A4是参与GA 8 - 羟基化的主要同工酶。重组人CYP3A4和人肝微粒体中的GA 8 - 羟基化均遵循经典的米氏动力学。内在清除率数值表明，CYP3A4对GA 8 - 羟基化的贡献比CYP3A5高12.8倍。分子对接研究表明，CYP3A4与CYP3A5之间存在不同的氢键和π - π相互作用，这可能导致它们对GA 8 - 羟基化的催化活性不同。此外，GA对CYP3A4的抑制活性强于对CYP3A5的抑制活性，这进一步表明CYP3A4对GA的转化具有更高的选择性。更重要的是，GA已成功应用于选择性监测诱导剂利福平对HepG2细胞中CYP3A4活性的调节，这与CYP3A4 mRNA表达水平的变化一致。总之，我们的研究结果表明，GA可作为一种新型探针，用于在组织和细胞制剂中选择性检测CYP3A4。
Nearly half of prescription medicines are metabolized by human cytochrome P450 (CYP) 3A. CYP3A4 and 3A5 are two major isoforms of human CYP3A and share most of the substrate spectrum. A very limited previous study distinguished the activity of CYP3A4 and CYP3A5, identifying the challenge in predicting CYP3A/mediated drug clearance and drug-drug interaction. In the present study, we introduced gomisin A (GA) with a dibenzocyclooctadiene skeleton as a novel selective probe of CYP3A4. The major metabolite of GA was fully characterized as 8-hydroxylated GA by LC-MS and NMR. CYP3A4 was assigned as the predominant isozyme involved in GA 8-hydroxylation by reaction phenotyping assays, chemical inhibition assays, and correlation studies. GA 8-hydroxylation in both recombinant human CYP3A4 and human liver microsomes followed classic Michaelis-Menten kinetics. The intrinsic clearance values indicated that CYP3A4 contributed 12.8-fold more than CYP3A5 to GA 8-hydroxylation. Molecular docking studies indicated different hydrogen bonds and pi-pi interactions between CYP3A4 and CYP3A5, which might result in the different catalytic activity for GA 8-hydroxylation. Furthermore, GA exhibited a stronger inhibitory activity towards CYP3A4 than CYP3A5, which further suggested a preferred selectivity of CYP3A4 for the transformation of GA. More importantly, GA has been successfully applied to selectively monitor the modulation of CYP3A4 activities by the inducer rifampin in hepG2 cells, which is consistent with the level change of CYP3A4 mRNA expression. In summary, our results suggested that GA could be used as a novel probe for the selective sensing of CYP3A4 in tissue and cell preparations.