Vitae Pharmaceuticals, 502 West Office Center Drive, Fort Washington, PA 19034, USA.
Chem Res Toxicol. 2010 Aug 16;23(8):1393-404. doi: 10.1021/tx100141d.
The in vitro bioactivation of the selective serotonin and norepinephrine reuptake inhibitor duloxetine was investigated using liver microsomes and cytosol, expressed glutathione transferase, and recombinant P450 2D6 and 1A2. In the presence of glutathione, several conjugates were identified and characterized using a combination of direct infusion nanoelectrospray mass spectrometry on an LTQ/Orbitrap and liquid-chromatography mass spectrometry on a triple quadrupole. Structural characterization of these conjugates revealed that glutathione conjugation occurred on naphthalene rather than on thiophene and likely proceeded via a reactive epoxide intermediate. Experiments with recombinant P450s and the isoform specific inhibitors quinidine and furafylline suggested that both P450 2D6 and 1A2 were involved in the bioactivation of duloxetine. To explore the utility of in silico approaches to address bioactivation issues, MetaSite and two docking approaches (rigid and induced-fit docking) utilizing publicly available human P450 crystal structures or a homology model for P450 2C19 were used to predict the sites of bioactivation for duloxetine as well as the thiophene containing compounds tienilic acid, suprofen, ticlopidine, methapyrilene, and OSI-930 for which glutathione conjugates on the thiophene moiety have been reported. MetaSite and induced fit docking but not rigid docking correctly predicted that naphthalene rather than thiophene was the preferred site of bioactivation for duloxetine by P450 2D6. MetaSite predictions were also consistent with literature reports that thiophene was the site of glutathione conjugation for tienilic acid, suprofen, and OSI-930 but not for ticlopidine or methapyrilene. Of the two docking approaches investigated, induced fit docking results were consistent with thiophene as the site of bioactivation for all compounds to which it was applied. In conclusion, our investigation identified the likely bioactivation pathway for duloxetine and demonstrated the utility of in silico approaches MetaSite and induced fit docking to address potential bioactivation liabilities.
我们使用肝微粒体和细胞液、表达的谷胱甘肽转移酶以及重组 P450 2D6 和 1A2 研究了选择性 5-羟色胺和去甲肾上腺素再摄取抑制剂度洛西汀的体外生物活化。在谷胱甘肽存在的情况下,使用直接注入纳升电喷雾质谱法在 LTQ/Orbitrap 和三重四极杆上的液相色谱质谱联用,鉴定并表征了几种缀合物。这些缀合物的结构特征表明,谷胱甘肽与萘而非噻吩结合,可能通过反应性环氧化物中间体进行。使用重组 P450 以及同工型特异性抑制剂奎尼丁和呋喃西林的实验表明,P450 2D6 和 1A2 均参与了度洛西汀的生物活化。为了探索使用计算方法解决生物活化问题的实用性,MetaSite 以及两种对接方法(刚性对接和诱导契合对接),利用公共的人类 P450 晶体结构或 P450 2C19 的同源模型,用于预测度洛西汀以及含有噻吩的化合物替比林酸、舒洛芬、噻氯匹定、美沙吡林和 OSI-930 的生物活化部位,这些化合物的噻吩部分已经报道了谷胱甘肽缀合物。MetaSite 和诱导契合对接但不是刚性对接正确地预测,萘而不是噻吩是 P450 2D6 生物活化度洛西汀的首选部位。MetaSite 预测也与文献报道一致,即噻吩是替比林酸、舒洛芬和 OSI-930 谷胱甘肽缀合的部位,但不是噻氯匹定或美沙吡林的部位。在所研究的两种对接方法中,诱导契合对接的结果与噻吩作为所有应用化合物的生物活化部位一致。总之,我们的研究确定了度洛西汀可能的生物活化途径,并证明了计算方法 MetaSite 和诱导契合对接在解决潜在生物活化问题方面的实用性。