【作者】 刘倩；

【导师】 Matthias BUREIK； 李格如；

【作者基本信息】 天津大学 ， 药学硕士（专业学位）， 2022， 硕士

【摘要】 细胞色素P450酶(CYPs或P450)是血红素蛋白的一个超家族,可以催化氧化还原反应。它们广泛存在于动物、植物、真菌和细菌中,在人体内存在57种CYP酶。除了存在于肝脏,P450酶还表达于许多其他组织,包括肠粘膜(可能是药物代谢最重要的肝外组织)、脑、肾、肺和皮肤。它们参与关键内源性物质的生物合成,以及内源性物质和外源性物质(包括药物和环境化合物)的代谢。CYP3A亚家族的含量约占肝脏内P450酶总含量的30%,而临床使用的药物中却有50%以上由该亚家族代谢。CYP3A亚家族有四个成员,分别命名为CYP3A4、CYP3A5、CYP3A7和CYP3A43。CYP3A4、CYP3A5、CYP3A7主要负责代谢外源性物质,而CYP3A43的药理作用目前还没有统一的定论,被认为是“孤儿”CYP酶。我们课题组之前通过使用渗透性裂变酵母对五种相同的前体荧光素物质进行生物转化,研究了57种人类细胞色素P450酶的相关性。结果表明,CYP3A4、CYP3A5和CYP3A7对这五种探针底物的代谢具有相似的偏好,而CYP3A43则与其他三种酶表现出不同的模式。为了进一步探究四个CYP3A亚家族成员之间的关系,在本研究中,我们使用了12种相同的候选抑制剂对CYP3A亚家族成员进行抑制试验。通过这种方式,我们构建了CYP3A亚家族的第一个抑制剂谱。在本试验中,我们发现了三种CYP3A4抑制剂、五种CYP3A5抑制剂、五种CYP3A7抑制剂和四种CYP3A43抑制剂。有趣的是,芳香化酶(CYP19A1)抑制剂来曲唑对CYP3A43表现出高选择性。对CYP3A亚家族的相关分析表明,CYP3A4和CYP3A7的抑制剂谱比CYP3A5或CYP3A43的抑制剂谱更相似。我们的合作伙伴Gerhard Wolber教授(德国柏林自由大学)通过使用CYP3A43的同源模型并进行抑制剂对接,帮助解释了来曲唑在分子水平上对CYP3A43的特殊选择性。他们还对CYP3A43模型中选定的来曲唑对接姿势进行了广泛的无约束全原子MD模拟。他们推测Thr374的羟基在稳定来曲唑方面起着重要作用,因为之前的研究已经概述了化学上足够的氢键受体(HBA)基团相对于来曲唑三唑在空间上适当定位的相关性。因此,Thr374和来曲唑氰基氮原子之间建立的氢键为来曲唑的抑制选择性提供了合理的解释。这一新发现的效应是否具有生理作用将在未来的研究中予以阐明。更多还原

【Abstract】 Cytochrome P450 enzymes(CYPs or P450s)are a superfamily of heme proteins that can catalyze redox reactions.They widely occur in animals,plants,fungi and bacteria,and there are 57 individual CYP enzymes existing in human beings.In addition to being present in the liver,P450 enzymes are also expressed in many other tissues,including intestinal mucosa(possibly the most important extrahepatic tissue for drug metabolism),brain,kidney,lung and skin.They participate in the biosynthesis of critical endogenous substances and the metabolism of both endogenous substances and exogenous substances including drugs and environmental compounds.Among the P450 enzymes in the liver,the CYP3 A subfamily accounts for about 30% of protein content,and more than 50% of drugs in clinical use are metabolized by this subfamily.There are four members in the CYP3 A subfamily,named CYP3A4,CYP3A5,CYP3A7 and CYP3A43.CYP3A4,CYP3A5,CYP3A7 were thought to be responsible for metabolism of xenobiotics,while CYP3A43 is an orphan CYP whose pharmaceutical effects were not clear so far.A previous study from our group was aimed at the correlation of the 57 human cytochrome P450 by using recombinant fission yeast strains to conduct biotransformations of five proluciferin esters.It was found that CYP3A4,CYP3A5 and CYP3A7 have similar preferences on metabolizing these five probe substrates,while CYP3A43 showed a different pattern.In order to future explore the relationship between the four CYP3 A subfamily members,in this study we used twelve candidate inhibitor compounds to perform inhibition assays.In this way we constructed the first inhibitor profile of the CYP3 A subfamily.During these experiments,three inhibitors of CYP3A4,five inhibitors of CYP3A5,five inhibitors of CYP3A7,and four inhibitors of CYP3A43 were found.Interestingly,the aromatase(CYP19A1)inhibitor letrozole showed high selectivity towards CYP3A43.A correlation analysis of CYP3 A subfamily showed that the inhibitor profiles of CYP3A4 and CYP3A7 are more similar to each other than those of CYP3A5 or CYP3A43.Our cooperation partner Prof.Gerhard Wolber(Free University Berlin,Germany)helped to explain the special selectivity of letrozole to CYP3A43 at the molecular level by using a homology model of CYP3A43 and performing inhibitor docking.They also performed extensive unconstrained all-atom MD simulations of the selected letrozole docking pose in our CYP3A43 model.they surmise that the hydroxyl group of Thr374 plays an important role in stabilizing letrozole,seeing as the relevance of chemically adequate hydrogen bond acceptor(HBA)groups positioned sterically appropriately relative to letrozole triazole has been outlined in previous research.Thus,the hydrogen bond established between Thr374 and letrozole cyano group nitrogen provides a plausible explanation for the inhibitory selectivity of letrozole.Whether this newly discovered effect is of physiological consequence will have to be elucidated in future studies.更多还原