THIOPHENE DERIVATIVES AS NEW MECHANISM-BASED INHIBITORS OF CYTOCHROME-P-450 - INACTIVATION OF YEAST-EXPRESSED HUMAN LIVER CYTOCHROME-P-450-2C9 BY TIENILIC ACID
THIOPHENE DERIVATIVES AS NEW MECHANISM-BASED INHIBITORS OF CYTOCHROME-P-450 - INACTIVATION OF YEAST-EXPRESSED HUMAN LIVER CYTOCHROME-P-450-2C9 BY TIENILIC ACID
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DOI：
10.1021/bi00167a022
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发表时间：
1994-01-11
期刊：
影响因子：
2.9
通讯作者：
MANSUY, D
中科院分区：
文献类型：
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作者：
LOPEZGARCIA, MP;DANSETTE, PM;MANSUY, D
通过表达两种密切相关的人肝细胞色素P-450（P450）2C 9和2C 10的酵母微粒体氧化替尼酸（TA），导致这些P450活性的催化依赖性丧失。在相同的条件下，噻苯酸异构体（TAI）的氧化不能使P45 O失活。TA氧化过程中P450活性的丧失伴随着产物（5-羟基噻吩甲酸，5-OHTA）的形成，表现出伪一级和饱和动力学，并被替代底物甲苯磺丁脲抑制。TA代谢产物与微粒体蛋白的共价结合与酶失活平行发生，并被反应介质中谷胱甘肽的存在部分抑制。然而，谷胱甘肽不能保护P450酶免于失活。因此，TA表现出P450 2C 9和2C 10酶的基于机制的灭活剂的所有特征。在P450 2C 10的情况下，测定了以下动力学参数：t1/2，max = 3.4 min，k（inact）= 3.6 × 10（-3）s-1，K（I）= 4.3 μ M，k（inact）/K（I）= 813 L mol-1 s-1，分配比= 11.6。此外，发现每摩尔P450 2C 10 0.9摩尔TA代谢物的特异性共价结合发生在酶活性完全丧失之前（在谷胱甘肽存在下进行的孵育中）。提出了TA氧化过程中P450 2C 10（2C 9）失活的合理机制。它涉及亲电噻吩亚砜的中间体形成，其可以在其噻吩环的5位与H2O反应以产生5-OHTA或与P450活性位点的氨基酸残基的亲核基团反应，这导致其与P450蛋白的共价结合。TA对P450 2C 9（2C 10）的烷基化和失活可能是在接受TA治疗的免疫过敏性肝炎患者中检测到抗P450 2C抗体的起点。
Oxidation of tienilic acid (TA) by microsomes of yeast expressing two closely related human liver cytochrome P-450s (P450), P450 2C9 and 2C10, led to catalysis-dependent loss of activity of these P450s. Under identical conditions, oxidation of a tienilic acid isomer (TAI) failed to give any P45O inactivation. The loss of P450 activity during TA oxidation was concomitant with product (5-hydroxytienilic acid, 5-OHTA) formation, showed pseudo-first-order and saturation kinetics, and was inhibited by an alternative substrate, tolbutamide. Covalent binding of TA metabolites to microsomal proteins occurred in parallel with enzyme inactivation and was partially inhibited by the presence of glutathione in the reaction medium. However, glutathione did not protect P450 enzyme from inactivation. Thus, TA exhibited all of the characteristics of a mechanism-based inactivator for P450 2C9 and 2C10 enzymes. The following kinetic parameters were determined in the case of P450 2C10: t1/2,max = 3.4 min, k(inact) = 3.6 10(-3) s-1, K(I) = 4.3 muM, k(inact)/K(I) = 813 L mol-1 s-1, and partition ratio = 11.6. Moreover, a specific covalent binding of 0.9 mol of TA metabolite per mole of P450 2C10 was found to occur before the complete loss of enzyme activity (in incubations performed in the presence of glutathione). A plausible mechanism for P450 2C10 (2C9) inactivation during TA oxidation is proposed. It involves the intermediate formation of an electrophilic thiophene sulfoxide, which may react at position 5 of its thiophene ring either with H2O to give 5-OHTA or with a nucleophilic group of an amino acid residue of the P450 active site, which results in its covalent binding to P450 protein. This alkylation and inactivation of P450 2C9 (2C10) by TA could be a starting point for the appearance of anti-P450 2C antibodies detected in patients treated with TA and suffering from immunoallergic hepatitis.