Herbacetin Broadly Blocks the Activities of CYP450s by Different Inhibitory Mechanisms
Herbacetin Broadly Blocks the Activities of CYP450s by Different Inhibitory Mechanisms
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DOI：
10.1055/a-1502-7131
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发表时间：
2021-06
期刊：
影响因子：
2.7
通讯作者：
Jianchang Qian;Ying‐hui Li;Xiaodan Zhang;Daoxing Chen;Mingming Han;Tao Xu;Bingbing Chen;G. Hu-G.
中科院分区：
文献类型：
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作者：
Jianchang Qian;Ying‐hui Li;Xiaodan Zhang;Daoxing Chen;Mingming Han;Tao Xu;Bingbing Chen;G. Hu-G.
摘要 Herbacetin 是一种具有生物活性的黄烷醇化合物，具有多种药理作用。然而，其药代动力学特征尚未得到彻底研究。此前，我们筛选了一个天然化合物库，并鉴定出 Herbacetin 是一种有效的 CYP 阻滞剂。在此，我们的目的是从机制上确定 Herbacetin 对 CYP450 的抑制作用及其潜在应用。基于UPLC-MS/MS方法开发了人肝微粒体孵育系统。此外，还开发了计算机对接测定和人 CYP 重组酶反应系统，并用于研究结合亲和力和抑制功效。随后，通过 MTT 和免疫印迹测定评估了 Herbacetin 和索拉非尼组合对 HepG2 细胞的影响。在有或没有赫巴西汀孵育后，通过 UPLC-MS/MS 测量索拉非尼及其主要代谢物的浓度。结果，我们发现 100 µM 的草苷几乎完全抑制主要 CYP 的功能。此外，通过分析构效关系，我们发现4-、6-和8-羟基是抑制作用的重要基团。 Herbacetin 以混合方式抑制 CYP3A4、CYP2B6、CYP2C9 和 CYP2E1，但非竞争性阻断 CYP2D6。这些结果与体外重组酶反应结果非常一致，每种测试同工酶的 IC50 < 10 µM。有趣的是，索拉非尼与赫巴西汀联合使用时，对 HepG2 细胞凋亡的刺激作用显着增强，这与索拉非尼暴露量的增加有关。总之，Herbacetin 是一种广谱 CYP450 的有效抑制剂，可能会增强药物在体内的暴露。
Abstract Herbacetin is a bioactive flavanol compound that has various pharmacological effects. However, the pharmacokinetic characteristics have not been thoroughly investigated. Previously, we screened a natural compound library and identified herbacetin as a potent CYP blocker. Herein, we aimed to mechanistically determine the inhibitory effects of herbacetin on CYP450 and its potential application. A human liver microsome incubation system was developed based on a UPLC-MS/MS method. Moreover, an in silico docking assay and a human CYP recombinase reaction system were developed and used to investigate binding affinity and inhibitory efficacy. Subsequently, the effects of the combination of herbacetin and sorafenib on HepG2 cells were assessed by MTT and immunoblotting assays. The concentration of sorafenib and its main metabolite were measured by UPLC-MS/MS after incubation with or without herbacetin. As a result, we found herbacetin almost completely inhibited the functions of major CYPs at 100 µM. Moreover, through analysis of the structure-activity relationship, we found 4-, 6-, and 8-hydroxyl were essential groups for the inhibitory effects. Herbacetin inhibited CYP3A4, CYP2B6, CYP2C9, and CYP2E1 in a mixed manner, but non-competitively blocked CYP2D6. These results are in good agreement with the recombinase reaction in vitro results, with an IC50 < 10 µM for each tested isoenzyme. Interestingly, the stimulatory effects of sorafenib on HepG2 cell apoptosis were significantly enhanced by combining with herbacetin, which was associated with increased sorafenib exposure. In summary, herbacetin is a potent inhibitor of a wide spectrum of CYP450s, which may enhance the exposure of drugs in vivo.