Mancy A, Broto P, Dijols S, Dansette P M, Mansuy D
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, URA 400 CNRS, Université Paris V, France.
Biochemistry. 1995 Aug 22;34(33):10365-75. doi: 10.1021/bi00033a007.
Biochemical experiments, using the well-defined human liver CYP2C9 expressed in yeast, and molecular modeling techniques were used to derive a predictive model for substrates of CYP2C9. The ability of 10 2-aroylthiophenes related to tienilic acid to act as substrates for CYP2C9 was studied. Four of them were original compounds that were synthesized and completely characterized by several spectroscopic techniques. In these 10 compounds various chemical functions, such as ester, amide, alcohol, phenol, ether or tetrazole functions, replaced the OCH2COOH function of tienilic acid. Among them, only the derivatives containing an acidic function (carboxylic acids, phenol, and tetrazole whose pKaS are 4.8, 6.3, and 3.8, respectively) underwent a 5-hydroxylation of their thiophene ring like tienilic acid. Despite their close structural analogy with tienilic acid, all of the other compounds not only did not undergo any 5-hydroxylation of their thiophene ring but also failed to act as inhibitors of CYP2C9. These results strongly suggested that the presence, at pH 7.4, of a negative charge on the substrate is a very important feature in its recognition by CYP2C9. In fact, the four new substrates of CYP2C9 described in this study, a carboxylic acid, phenol, and tetrazole derivative, each of which is related to tienilic acid, and the antiinflammatory drug, suprofen (with Km between 12 and 130 microM and kcat between 0.2 and 1.3 min-1), as well as almost all CYP2C9 substrates reported in the literature, exhibit a pKa below 7 (except phenytoin whose pKa is 8.1). They mainly exist as anions at physiological pH. By using molecular modeling techniques, 12 CYP2C9 substrates were superimposed with respect to their hydroxylation site and fitted onto templates, which were rigid molecules such as (S)-warfarin and phenytoin. It was thus possible to arrange them in order that all their anionic sites were at a distance around 4 A from a common point (a putative cationic site of the protein) in space. These results provide a model of the substrate binding site of CYP2C9, in which substrates interact through their anionic site A- with a cationic residue of the CYP2C9 protein C+. In that model, the distance between the hydroxylation site (Hy) and the anionic site (A-) is 7.8 +/- 1.6 A, and the <HyA-C+ angle is 82 +/- 15 degrees.
利用在酵母中表达的纯化的人肝脏CYP2C9进行生化实验，并结合分子建模技术，推导出了CYP2C9底物的预测模型。研究了10种与替尼酸相关的2-芳酰基噻吩作为CYP2C9底物的能力。其中4种是通过几种光谱技术合成并完全表征的原始化合物。在这10种化合物中，各种化学官能团，如酯、酰胺、醇、酚、醚或四唑官能团，取代了替尼酸的OCH2COOH官能团。其中，只有含有酸性官能团（羧酸、苯酚和四唑，其pKa分别为4.8、6.3和3.8）的衍生物像替尼酸一样在其噻吩环上发生5-羟基化反应。尽管它们与替尼酸结构相似，但所有其他化合物不仅没有在其噻吩环上发生任何5-羟基化反应，而且也没有作为CYP2C9的抑制剂。这些结果强烈表明，在pH 7.4时底物上存在负电荷是其被CYP2C9识别的一个非常重要的特征。事实上，本研究中描述的CYP2C9的四种新底物，一种羧酸、苯酚和四唑衍生物，它们各自都与替尼酸有关，以及抗炎药舒洛芬（Km在12至130 microM之间，kcat在0.2至1.3 min-1之间），以及文献中报道的几乎所有CYP2C9底物，其pKa均低于7（除苯妥英钠的pKa为8.1外）。它们在生理pH下主要以阴离子形式存在。通过分子建模技术，将12种CYP2C9底物相对于它们的羟基化位点进行叠加，并拟合到模板上，这些模板是刚性分子，如（S）-华法林和苯妥英钠。因此，可以将它们排列成这样，即它们所有的阴离子位点在空间中距离一个共同点（蛋白质的一个假定阳离子位点）约4埃。这些结果提供了一个CYP2C9底物结合位点的模型，其中底物通过其阴离子位点A-与CYP2C9蛋白C+的一个阳离子残基相互作用。在该模型中，羟基化位点（Hy）与阴离子位点（A-）之间的距离为7.8±1.6埃，<HyA-C+角为82±15度。
