Ha-Duong N T, Dijols S, Macherey A C, Goldstein J A, Dansette P M, Mansuy D
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris V, 45 Rue des Saints-Pères, 75270 Paris Cedex 06, France.
Biochemistry. 2001 Oct 9;40(40):12112-22. doi: 10.1021/bi010254c.
Experiments using recombinant yeast-expressed human liver cytochromes P450 confirmed previous literature data indicating that ticlopidine is an inhibitor of CYP 2C19. The present studies demonstrated that ticlopidine is selective for CYP 2C19 within the CYP 2C subfamily. UV-visible studies on the interaction of a series of ticlopidine derivatives with CYP 2C19 showed that ticlopidine binds to the CYP 2C19 active site with a K(s) value of 2.8 +/- 1 microM. Derivatives that do not involve either the o-chlorophenyl substituent, the free tertiary amine function, or the thiophene ring of ticlopidine did not lead to such spectral interactions and failed to inhibit CYP 2C19. Ticlopidine is oxidized by CYP 2C19 with formation of two major metabolites, the keto tautomer of 2-hydroxyticlopidine (1) and the dimers of ticlopidine S-oxide (TSOD) (V(max) = 13 +/- 2 and 0.4 +/- 0.1 min(-1)). During this oxidation, CYP 2C19 was inactivated; the rate of its inactivation was time and ticlopidine concentration dependent. This process meets the chemical and kinetic criteria generally accepted for mechanism-based enzyme inactivation. It occurs in parralel with CYP 2C19-catalyzed oxidation of ticlopidine, is inhibited by an alternative well-known substrate of CYP 2C19, omeprazole, and correlates with the covalent binding of ticlopidine metabolite(s) to proteins. Moreover, CYP 2C19 inactivation is not inhibited by the presence of 5 mM glutathione, suggesting that it is due to an alkylation occurring inside the CYP 2C19 active site. The effects of ticlopidine on CYP 2C19 are very analogous with those previously described for the inactivation of CYP 2C9 by tienilic acid. This suggests that a similar electrophilic intermediate, possibly a thiophene S-oxide, is involved in the inactivation of CYP 2C19 and CYP 2C9 by ticlopidine and tienilic acid, respectively. The kinetic parameters calculated for ticlopidine-dependent inactivation of CYP 2C19, i.e., t(1/2max) = 3.4 min, k(inact) = 3.2 10(-3) s(-1), K(I) = 87 microM, k(inact)/K(I) = 37 L.mol(-1).s(-1), and r (partition ratio) = 26 (in relation with formation of 1 + TSOD), classify ticlopidine as an efficient mechanism-based inhibitor although somewhat less efficient than tienilic acid for CYP 2C9. Importantly, ticlopidine is the first selective mechanism-based inhibitor of human liver CYP 2C19 and should be a new interesting tool for studying the topology of the active site of CYP 2C19.
使用重组酵母表达的人肝细胞色素P450进行的实验证实了先前文献数据，表明噻氯匹定是CYP 2C19的抑制剂。目前的研究表明，噻氯匹定在CYP 2C亚家族中对CYP 2C19具有选择性。对一系列噻氯匹定衍生物与CYP 2C19相互作用的紫外可见研究表明，噻氯匹定以2.8±1 microM的K(s)值与CYP 2C19活性位点结合。不涉及噻氯匹定的邻氯苯基取代基、游离叔胺官能团或噻吩环的衍生物不会导致这种光谱相互作用，也无法抑制CYP 2C19。噻氯匹定被CYP 2C19氧化，形成两种主要代谢物，即2-羟基噻氯匹定的酮式互变异构体(1)和噻氯匹定S-氧化物(TSOD)的二聚体(V(max)=13±2和0.4±0.1 min(-1))。在这种氧化过程中，CYP 2C19失活；其失活速率与时间和噻氯匹定浓度有关。这个过程符合基于机制的酶失活普遍接受的化学和动力学标准。它与CYP 2C19催化的噻氯匹定氧化同时发生，受到CYP 2C19的另一种著名底物奥美拉唑的抑制，并且与噻氯匹定代谢物与蛋白质的共价结合相关。此外，5 mM谷胱甘肽的存在不会抑制CYP 2C19失活，这表明它是由于在CYP 2C19活性位点内发生的烷基化。噻氯匹定对CYP 2C19的作用与先前描述的替尼酸使CYP 2C9失活的作用非常相似。这表明，分别参与噻氯匹定和替尼酸使CYP 2C19和CYP 2C9失活的可能是类似的亲电中间体，可能是噻吩S-氧化物。为噻氯匹定依赖性CYP 2C19失活计算的动力学参数，即t(1/2max)=3.4分钟，k(inact)=3.2×10(-3)s(-1)，K(I)=87 microM，k(inact)/K(I)=37 L·mol(-1)·s(-1)，以及r(分配比)=26(与1+TSOD的形成有关)，将噻氯匹定归类为一种有效的基于机制的抑制剂，尽管对于CYP 2C9来说，其效率略低于替尼酸。重要的是，噻氯匹定是人类肝脏CYP 2C19的首个选择性基于机制的抑制剂，应该是研究CYP 2C19活性位点拓扑结构的一个新的有趣工具。
