Mechanism-based inactivation of cytochrome P450 2C9 by tienilic acid and (+/-)-suprofen: a comparison of kinetics and probe substrate selection.
Mechanism-based inactivation of cytochrome P450 2C9 by tienilic acid and (+/-)-suprofen: a comparison of kinetics and probe substrate selection.
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DOI：
10.1124/dmd.108.023358
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发表时间：
2009-01
影响因子：
3.9
通讯作者：
Tracy, Timothy S.
中科院分区：
文献类型：
--
作者：
Hutzler, J. Matthew;Balogh, Larissa M.;Zientek, Michael;Kumar, Vikas;Tracy, Timothy S.
采用（S）-氟比洛芬、双氯芬酸和（S）-华法林作为报告底物，进行了体外实验，以比较噻尼酸和（±）-舒洛芬对P450 2C 9的kinact、KI和灭活效率（kinact/KI）。当使用（S）-氟比洛芬和双氯芬酸作为底物时，噻尼酸对P450 2C 9的灭活相似（效率为1.9 ml/min/μmol），灭活动力学特征为S形曲线。（±）-舒洛芬对（S）-氟比洛芬和双氯芬酸羟基化的失活也通过S形曲线描述，尽管失活效率明显较低（<1 ml/min/μmol）。相反，由替尼酸和（±）-舒洛芬介导的P450 2C 9的（S）-华法林7-羟基化的失活最符合双曲线方程，其中失活效率适度较高（10 ml/min/μmol），且高出1.3倍（3 ml/min/μmol），相对于其他探针底物，这表明在体外评估基于机制的P450 2C 9失活时应仔细考虑报告底物。对观察到的噻尼酸相对于（±）-舒洛芬的灭活增加进行的进一步研究表明，噻尼酸是一种更高亲和力的底物，光谱结合亲和力常数（Ks）为2 μM，体外半衰期为5 min，而（±）-舒洛芬的Ks为21 μM，体外半衰期为50 min。最后，具有被环氧乙烷环取代的羧酸酯官能团的噻尼酸的紧密类似物没有失活特性，这表明与P450 2C 9活性位点中的带正电荷的残基的离子结合相互作用对于这些紧密结构类似物的识别和基于机制的失活是至关重要的。
In vitro experiments were conducted to compare kinact, KI and inactivation efficiency (kinact/KI) of P450 2C9 by tienilic acid and (±)-suprofen using (S)-flurbiprofen, diclofenac, and (S)-warfarin as reporter substrates. While the inactivation of P450 2C9 by tienilic acid when using (S)-flurbiprofen and diclofenac as substrates was similar (efficiency of ∼9 ml/min/μmol), the inactivation kinetics were characterized by a sigmoidal profile. (±)-Suprofen inactivation of (S)-flurbiprofen and diclofenac hydroxylation was also described by a sigmoidal profile, although inactivation was markedly less efficient (∼1 ml/min/μmol). In contrast, inactivation of P450 2C9 mediated (S)-warfarin 7-hydroxylation by tienilic acid and (±)-suprofen was best fit to a hyperbolic equation, where inactivation efficiency was moderately higher (10 ml/min/μmol), and ∼3-fold higher (3 ml/min/μmol), respectively, relative to the other probe substrates, which argues for careful consideration of reporter substrate when assessing mechanism-based inactivation of P450 2C9 in vitro. Further investigations into the observed increased inactivation by tienilic acid relative to (±)-suprofen revealed that it is a higher affinity substrate, with a spectral binding affinity constant (Ks) of 2 μM and an in vitro half-life of 5 min, compared to a Ks of 21 μM and a 50 min in vitro half-life for (±)-suprofen. Lastly, a close analog of tienilic acid with the carboxylate functionality replaced by an oxirane ring, was devoid of inactivation properties, which suggests that an ionic binding interaction with a positively-charged residue in the P450 2C9 active site is critical for recognition and mechanism-based inactivation by these close structural analogs.
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影响因子：
2.9
作者：
LOPEZGARCIA, MP;DANSETTE, PM;MANSUY, D
通讯作者：
MANSUY, D
影响因子：
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作者：
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通讯作者：
Venkatakrishnan, Karthik
影响因子：
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作者：
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通讯作者：
Backes, Wayne L.
影响因子：
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作者：
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通讯作者：
Goldstein, JA
影响因子：
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作者：
LECOEUR, S;BONIERBALE, E;BEAUNE, PH
通讯作者：
BEAUNE, PH