人肝细胞色素P450 2C金属酶与药物代谢*

由于人肝细胞色素P450 2C亚家族与临床药物代谢的密切关系,其研究已引起人们的广泛关注。本文综述了四种人肝细胞色素P450 2C,着重综述了其中的三种：CYP2C9,CYP2C8,CYP2C19的研究进展。评述了CYP2C9,CYP2C8和CYP2C19的某些氨基酸残基在催化过程中的作用,这三种酶的基因多态在不同人种中的分布及药物代谢的差异,以及它们与用药的特异性及某些疾病的易感性的联系,介绍了目前提出的CYP2C8的底物药效团模型,最后总结了CYP2C9,CYP2C8,CYP2C19,CYP2C18的主要特性。     

细胞色素P450对底物的选择性及催化机制

细胞色素P450酶是广泛存在的含亚铁血红素单加氧酶,参与甾类激素的合成、脂溶性维生素代谢、多不饱和脂肪酸转换为生物活性分子,以及致癌作用和药物代谢.综述了细胞色素P450结构与功能的关系,特别是细胞色素P450对底物的选择性以及催化机制,并对其未来的发展方向进行了展望.     

细胞色素p450的结构与催化机理

细胞色素P450酶是广泛存在的含亚铁血红素单加氧酶, 参与甾类激素的合成、脂溶性维生素代谢、多不饱和脂肪酸转换为生物活性分子, 以及致癌作用和药物代谢. 综述了细胞色素p450结构与功能的关系, 特别是细胞色素P450活性位点经历大幅度开/关运动结合底物和释放产物以及电子迁移途径.     

Rif-orf13编码的细胞色素P450催化利福霉素生物合成过程中C34a位的羟化反应（英文）

利福霉素生物合成途径在经历了二十余年的研究之后,仍然没有得到完全阐明.其中C34a甲基的氧化脱除是利福霉素成熟过程中的必需反应步骤,但是催化这一步骤的酶尚未鉴定;推测可能是利福霉素生物合成基因簇编码的某个细胞色素P450催化了这一步骤.选取利福霉素生物合成基因簇中功能尚未确证的P450基因rif-orf0、rif-orf4和rif-orf13在变铅青链霉菌中进行异源表达和底物喂养实验,发现表达了rif-orf13的链霉菌能够将16-脱甲基-34a-脱氧利福霉素W (1)转化为16-脱甲基利福霉素W (2).将rif-orf13在大肠杆菌BL21 (DE3)中进行诱导表达,利用纯化的Orf13蛋白进行体外酶催化反应,发现Orf13能够将底物1羟化为产物2.结合前人的基因敲除研究,认为rif-orf13是编码34a-脱氧利福霉素W羟化酶的基因,其在胞内的功能可以被另一个负责C12-C29双键氧化断裂的P450基因rif-orf5替代.     

液相色谱-质谱联用技术结合多探针底物法研究人参皂苷Rb1的体外代谢

以奥美拉唑、 苯妥英、 卡马西平和非那西丁为检测肝药酶细胞色素P450酶(CYP450)亚型的专属探针药物, 通过原型药物减少量测定法考察药物体外代谢的变化, 评价人参皂苷Rb1对CYP450不同亚型酶的作用. 结果表明, P2C9, P2C19和P3A4实验组与对照组差异不显著, P1A2实验组与对照组差异显著, 表明人参皂苷Rb1能诱导P1A2亚型酶的活性, 促进底物与酶反应, 加快底物的代谢, 而对P2C9, P2C19和P3A4三个亚型酶有弱的诱导或无诱导作用. 根据快速分离液相色谱-质谱联用(RRLC-MS/MS)检测结果推断, 人参皂苷Rb1在CYP450酶中的代谢产物可转化为人参皂苷Rb1氧化产物(Rb1+O)及人参皂苷Rd和F2.     

细胞色素C的光谱研究

细胞色素C是吸呼链的1个重要组成部分,位于细胞色素C₁和细胞色素a之间,血红素辅基中的铁原子可交替地处于+3或+2氧化态^[1]。Smith^[2]和Osheroff等^[3]对细胞色素C与细胞色素C₁及a的结合进行了详细的研究。关于细胞色素C与小分子的相互作用,除Corthesy^[4]进行了与ATP的作用,Osheroff^[3]进行了与碳酸根的作用外,与其它小分子的作用以及氧化型、还原型之间的相互转化受介质的影响还未见报道。     

己烯雌酚与CYP2C9的计算模拟及多光谱法研究

为了研究己烯雌酚与CYP2C9的结合作用机制,结合分子模拟,荧光光谱等光谱实验多角度分析了两者的结合情况。首先从分子对接得到己烯雌酚与CYP2C9相互作用的最佳结合构象,然后通过动力学模拟研究了己烯雌酚与CYP2C9的复合物的稳定性以及构象变化,最后用多光谱法进行实验印证。对接结果表明己烯雌酚与CYP2C9反应可以自发进行,动力学模拟结果表明两者具有较强的结合能力。同时,荧光光谱实验得出两者的结合机制为静态猝灭且形成一个结合位点,热力学参数证明两者结合作用为疏水作用力;紫外光谱实验进一步证明两者结合后的CYP2C9的构象和周围环境发生改变。通过拟合分析酰胺I带,红外定量分析结果表明,与己烯雌酚作用后的CYP2C9蛋白质的二级结构发生改变。综上结果表明CYP2C9与己烯雌酚可以发生相互作用,理论与实验进行相互印证,为进一步研究CYP2C9的催化代谢机制提供参考。     

细胞色素C在单糖修饰金电极上的直接电化学

Hill等发现在4,4′-联吡啶存在时,细胞色素C在金电极上能进行准可逆的电化学反应。在研究细胞色素C的直接电化学过程中,人们又发现一些生物小分子如氨基酸、嘌呤等对细胞色素C的电化学反应有促进作用,但迄今未见有关糖类分子对细胞色素C电化学反应促进作用研究的报道。本文研究了5种单糖对细胞色素C电化学反应的促进作用。     

细胞色素P450酶在生物合成及有机合成中的催化功能及其应用

细胞色素P450酶分布广泛,主要参与生物体外源物质代谢与天然产物生物合成,能以结构多样的有机化合物作为底物催化多种类型的化学反应.P450酶可在温和条件下实现底物分子中C—H键的选择性氧化,因而在精细化学品、化学中间体及药物分子的生产上具有很高的实用价值及多年的应用历史.随着蛋白质工程、氧化还原伴侣工程、底物工程、代谢工程与合成生物学的发展,目前已可初步实现根据反应需求来理性设计或定向进化改造P450酶催化系统来高效催化多种有机反应,拓宽了P450酶在生物合成与有机合成反应中的应用范围.总结了近年来由细胞色素P450酶参与催化的主要反应类型,归纳了拓宽P450酶催化反应类型、提高催化活性和选择性的一些重要策略,并对未来P450酶在生物合成及有机合成反应中的应用发展前景和挑战进行了展望.     

细胞色素P450的电化学研究进展

细胞色素P450的电化学研究从一个侧面反映了为使细胞色素P450达到工业催化剂的最终目的人们所作的不懈努力。本文从细胞色素P450在电极上的电子转移研究,隧道扫描显微镜的微观成像研究和使用电极作为细胞色素P450的电子给体从而实现细胞色素P450底物转化三方面,评述了近年来细胞色素P450的电化学研究进展。     

Structural and functional insights into CYP2C8.3: A genetic polymorph of cytochrome P450 2C8

The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver, and carries out the oxidative metabolism of at least 5% of clinical drugs. Polymorphisms in CYP2C8 have been closely implicated in individualized medication. CYP2C8.3, a common polymorph of CYP2C8 with dual amino acid substitutions R139K and K399R, is found primarily in Caucasians. In this study, CYP2C8.3 and its wild type (WT) CYP2C8 were expressed in E. coli, and their purified proteins were characterized by UV-visible spectroscopy, mass spectrometry, and circular dichroism. Their thermal stability, substrate binding ability, and metabolic activity against paclitaxel were investigated. The electron transfer kinetics during paclitaxel metabolism by WT CYP2C8 or CYP2C8.3 was studied by stopped-flow kinetics. The results revealed that mutations in CYP2C8.3 did not greatly influence the heme active site or protein thermal stability and paclitaxel binding ability, but the metabolic activity against paclitaxel was significantly depressed to just 11% of that of WT CYP2C8. Electron transfer from CYP reductase to CYP2C8.3 was found to be significantly slower than that to WT CYP2C8 during catalysis, and this might be the main reason for the depressed metabolic activity. Since the polymorph CYP2C8.3 is defective in catalyzing substrates of CYP2C8 in vitro, it might be expected to have important clinical and pathophysiological consequences in homozygous individuals, and this study provides valuable information in this aspect.     

Cytochrome P450 2C9 polymorphism: Effect of amino acid substitutions on protein flexibility in the presence of tamoxifen

Tamoxifen is a prodrug and cytochrome P450 2C9 (CYP2C9) has a significant role in the formation of a therapeutically more potent metabolite (4-hydroxytamoxifen) than tamoxifen. Since CYP2C9 exhibits genetic polymorphism, it may contribute to different phenotypic drug response. Moreover, it may be misleading if the possibility of heterogeneous clinical observations of pharmacogenetic investigations is ignored. Above all, clinical investigation of all the polymorphic variants is beyond the scope of a pharmacogenetic study. Therefore, in order to understand the genotype-phenotype association, it is aimed to study the interatomic interactions of amino acid substitutions in CYP2C9 variants in the presence of tamoxifen. Computational structural biology approach was adopted to study the effect of amino acid substitutions of polymorphic variants of CYP2C9 R144C (*2), I359 L (*3), D360E (*5), R150H (*8), R335W (*11) and L90 P (*13) on the flexibility of the enzyme in the presence of tamoxifen. The mutations were selected based on previously determined associations on genotype and clinical outcome of drugs.Against the above plane, docking of tamoxifen was performed with the crystal structure representing the wild-type form of the enzyme. The docked conformation of tamoxifen was favourable for 4-hydroxylation with the site of metabolism within 5 Å of oxyferrylheme consistent with the drug metabolism pathway of tamoxifen. Further, the effect of amino acid substitutions CYP2C9 variants on the protein flexibility in the presence of tamoxifen in 4-hydroxy orientation was evaluated by molecular dynamics (MD) simulations.Distinct protein flexibility modulations between variants were observed in F/G segment constituting the substrate access/egress channels, helix B' involved with substrate specificity and helix I associated with the holding of substrates. Root Mean Square Fluctuation analysis of the trajectories of variants exhibited fluctuations in F/G segment, B’ and I helix. Dominant motions in the structure were identified by performing Principal Component Analysis on trajectories and the porcupine plot depicted displaced F/G segment in variants.Thus, the interatomic interaction study of CYP2C9 variants in the presence of tamoxifen predicts the plausible effect of the investigated variants on the therapeutic outcome of tamoxifen. It is presumed that the observations of the study would be meaningful to understand tamoxifen pharmacogenetics.     

Effective virtual screening protocol for CYP2C9 ligands using a screening site constructed from flurbiprofen and S-warfarin pockets

An effective virtual screening protocol was developed against an extended active site of CYP2C9, which was derived from X-ray structures complexed with flubiprofen and S-warfarin. Virtual screening has been effectively supported by our structure-based pharmacophore model. Importance of hot residues identified by mutation data and structural analysis was first estimated in an enrichment study. Key role of Arg108 and Phe114 in ligand binding was also underlined. Our screening protocol successfully identified 76% of known CYP2C9 ligands in the top 1% of the ranked database resulting 76-fold enrichment relative to random situation. Relevance of the protocol was further confirmed in selectivity studies, when 89% of CYP2C9 ligands were retrieved from a mixture of CYP2C9 and CYP2C8 ligands, while only 22% of CYP2C8 ligands were found applying the structure-based pharmacophore constraints. Moderate discrimination of CYP2C9 ligands from CYP2C18 and CYP2C19 ligands could also be achieved extending the application domain of our virtual screening protocol for the entire CYP2C family. Our findings further demonstrate the existence of an active site comprising of at least two binding pockets and strengthens the need of involvement of protein flexibility in virtual screening.     

Human Cytochrome P450 (CYP1A2)‐dsDNA Interaction in situ Evaluation Using a dsDNA‐electrochemical Biosensor

Human cytochrome CYP1A2 is one of the major hepatic cytochrome P450s involved in many drugs metabolism, and chemical carcinogens activation. The CYP1A2‐dsDNA interaction in situ evaluation using a DNA‐electrochemical biosensor and differential pulse voltammetry was investigated. A dsDNA‐electrochemical biosensor showed that CYP1A2 interacted with dsDNA causing conformational changes in the double helix chain and DNA oxidative damage. A preferential interaction between the dsDNA guanosine residues and CYP1A2 was found, as free guanine and 8‐oxoguanine, a DNA oxidative damage biomarker, oxidation peaks were detected. This was confirmed using guanine and adenine homopolynucleotides‐electrochemical biosensors. The CYP1A2‐dsDNA interaction and dsDNA conformation changes was also confirmed by UV‐Vis spectrophotometry.     

Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP‐HPLC method to serve as activity marker

In this study, a simple and reliable reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated to analyze S‐mephenytoin 4‐hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co‐expressing CYP2C19 and NADPH‐CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP‐HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP‐HPLC assay showed good linearity (r² = 1.00) with 4‐hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10^?2 μm . Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (K_m, V_max and K_i) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co‐expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP‐HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

Thermal decomposition of Pr[(C_5H_8NS_2)_3(C_(12)H_8N_2)]

The thermal behavior of the complex Pr[(C5H8NS2)3(C12H8N2)] in a dry nitrogen flow was examined by TG-DTG analysis. The TG-DTG investigations indicated that Pr[(C5H8NS2)3-(C12H8N2)] was decomposed into Pr2S3 and deposited carbon in one step where Pr2S3 predominated in the final products. The results of non-isothermal kinetic calculations showed that the decomposition stage was the random nucleation and subsequent growth mechanism (n = 2/3), the corresponding apparent activation energy ?was 115.89 kJ·mol-1 and the pre-expo-nential constant ln[A/s] was 7.8697. The empirical kinetics model equation was proposed as/(α) =3/2(1-α)[-ln(1-α)]1/3.The X-ray powder diffraction patterns of the thermal decomposition products at 800℃under N2 atmosphere show that the product can be indexed to the cubic Pr2S3 phase. The transmission electron microscopy (TEM) of the final product reveals the particle appearance of a diameter within 40 nm. The experimental results show that the praseodymium sulfide nanocrystal can be prepared from thermal decomposition of Pr[(C5H8NS2)3(C12H8N2)].     

Determinants of the Inhibition of DprE1 and CYP2C9 by Antitubercular Thiophenes

Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X‐ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1–TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB.     

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis–Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.