类黄酮抑制P糖蛋白的三维定量构效关系与作用模式

采用基于R基团搜索技术的Topomer CoMFA建立了30个类黄酮类P糖蛋白抑制剂的三维定量构效关系(3D-QSAR)模型, 并用包括9个样本的测试集验证模型的外部预测能力. 所得模型的拟合、 交互验证以及外部验证的复相关系数分别为r²=0.971, q²=0.728和\begin{array}{c}{r}_{\mathit{pred}}^2\end{array}=0.816. 在此基础上, 运用Surflex-dock分子对接法研究了白杨素及其异戊烯化衍生物与P糖蛋白的作用模式. 结果表明, 异戊烯化修饰可显著提高类黄酮的亲脂性, 修饰产物能更好地与P糖蛋白的疏水性口袋契合, 二者结合程度高.     

苯甲酸类化合物小鼠经口LD50定量构效关系的研究

采用化合物定量结构-毒性效应关系(QSTR)方法研究了39个苯甲酸类化合物的各种量化参数对小鼠经口急性毒性(LD50)的影响,并建立了毒性预测模型.为了得到更准确的毒性预测模型,各个参数的交叉项同时考虑在内,利用线性回归方法得到了苯甲酸类化合物小鼠经口急性毒性预测模型(相关系数为0.958 2).该模型具有很好的预测能力.     

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.     

皂甙的三维定量构效关系研究

针对目标分子柔性大的特点,在比较分子场分析(CoMFA)方法中采用交叉验证相关系数平方R^2引导的构象选择法。对12个皂甙分子的生物活性进行了三维定量构效关系研究。探讨了几种探针对构效关系结果的影响,并选择了一种较合理的“复合”探针方案。应用该复合探针构建CoMFA模型,发现影响药效的立体场与静电场的贡献分别为40%和40%,其它能量项的贡献为20%。该模型交叉验证的相关系数平方R^2为0.653,非交叉验证的R^2为0.991,方差比F(4,7)值130.195(即置信度99%以上),活性预计的标准偏差与极差比(s/△γ)为4.2%,表明模型具有较好的预测能力。根据该模型,预计在指定位置添加位阻较大的基团活性值提高将会比较明显。     

细胞色素P450酶电化学生物传感器的构建及其药物代谢应用

药物代谢过程是药物在体内产生药效和毒性的主要过程,发展廉价、方便、快速、高通量的体外药物代谢研究方法对新药的开发和设计、给药的方法和剂量、临床药物的检测等都有重要的指导意义. 细胞色素P450酶（CYP450酶）在药物的I相反应中起到关键作用,以电极代替辅酶NADPH提供CYP450酶催化反应过程中需要的两个电子,构建CYP450酶电化学生物传感器可实现药物的初步筛选. 大量研究表明,CYP450酶在电极表面合适的固定方法与电极材料可有效提高传感器的检测性能. 本文主要综述近年来CYP450酶电化学生物传感器的构建及其在药物代谢研究方面的应用,并展望其研发前景.     

超高效液相串联质谱法同时定量检测6个细胞色素P450酶探针代谢产物

应用超高效液相色谱-质谱联用技术(LC-MS/MS),建立了同时定量测定6个细胞色素P450酶(CYP)探针代谢产物的方法。用甲醇和乙腈混合溶剂沉淀肝微粒体孵育液中的蛋白,在ZORBAX-C18色谱柱(100 mm×4.6 mm,3.5μm)上,以5 mmol/L甲酸铵和0.1%甲酸-乙腈为流动相,梯度分离待测物。在串联质谱正离子多反应监测模式下定量检测待测物。方法学验证结果表明,6个代谢产物在1.0～1000.0μg/L的范围内均呈良好的线性关系(r2>0.994);定量限为1μg/L;方法的日内和日间精密度(RSD)均小于12%;加标回收率为92.8%～104.4%;不同储存条件下样品稳定性实验的浓度偏差(RSD)小于10%。人肝微粒体活性测定的结果显示CYP1A2和CYP3A4的酶活性最强,分别为(466.1±32.1)和(694.3±11.7)pmole/(mg.min),与最低的CYP2C19酶活性分别相差27.7和41.3倍。本方法简便、快速、灵敏,适用于大量化合物CYP酶诱导和抑制评价的酶活性测定。     

对氨基苯甲酸酯同系物的高效液相色谱保留行为与结构参数的相关性

测定了一组对氨基苯甲酸酯同系物的高效液相色谱容量因子ｋ′和保留指数ＩＲ,建立了ｌｎｋ′和ＩＲ与摩尔折射、ｖａｎｄｅｒＷａａｌｓ体积、一阶分子连接性指数、分子总能量和偶极矩等结构参数的相关方程式。     

氯苯胺类化合物对发光细菌毒性的定量构效关系

应用密度泛函理论的BLYP方法,对14种氯苯胺类化合物进行几何构型优化,得到稳定构型下的量子化学参数,研究了该类化合物对发光细菌毒性作用的定量结构-活性关系(QSAR),应用逐步回归方法建立了相关方程.结果表明:该类化合物对发光细菌的毒性作用随分子最低空轨道(LUMO)能级的降低而增大,随分子中氯取代数目的增加而增大.     

褪黑激素受体拮抗剂的三维定量构效关系研究

采用两种分子场分析方法即比较分子场分析法（CoMFA）和比较分子相似因子分析法（CoMSIA）进行了37个褪黑激素受体拮抗剂的构效关系研究.计算结果表明，两种方法得到的构效关系模型都具有较好的预测能力.在计算中，还考察了不同格点距离和电荷计算方法对构效关系模型的影响.通过分析分子场等值面图在空间的分布，可以观察到叠合分子周围分子场特征对化合物活性的影响，为设计新的褪黑激素拮抗剂提供了一些理论依据.     

Cytochrome P450 Database

Abstract

This paper describes a specialized database dedicated exclusively to the cytochrome P450 superfamily. The system provides the impression of superfamily's nomenclature and describes structure and function of different P450 enzymes. Information on P450-catalyzed reactions, substrate preferences, peculiarities of induction and inhibition is available through the database management system. Also the source genes and appropriate translated proteins can be retrieved together with corresponding literature references.

Developed programming solution provides the flexible interface for browsing, searching, grouping and reporting the information. Local version of database manager and required data files are distributed on a compact disk. Besides, there is a network version of the software available on Internet. The network version implies the original mechanism. which is useful for the permanent online extension of the data scope.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

Faradic Peaks Enhanced by Carbon Nanotubes in Microsomal Cytochrome P450 Electrodes

In this work we present an investigation on the behavior of microsomes containing human cytochrome P450 in cyclic voltammetry for drug detection. The microsomes are adsorbed on the surface of multi‐walled carbon nanotubes by drop‐casting. We demonstrate that the hydrophobic and highly electroactive surface of multi‐walled carbon nanotubes enables to distinguish more clearly the contributions in reduction peak current attributed to the enzymatic components of microsomes. Voltammetric measurements were performed under several experimental conditions with two cytochrome P450‐isoforms, 1A2 and 3A4. We show that the reduction current for the component of cytochrome P450‐microsome linearly increases in the presence of a substrate.     

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。     

Bacterial Cytochrome P450 Catalyzed Post-translational Macrocyclization of Ribosomal Peptides**

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450-catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence and co-conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450-catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp-Trp-Tyr (one C−C and two C−N bonds), Tyr-Trp (C−C bond), Trp-Trp (C−N bond), and His-His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization.     

Cytochrome P450 BM-3 Evolved by Random and Saturation Mutagenesis as an Effective Indole-Hydroxylating Catalyst

Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate. Three mutants (K434R/3X, E435D/3X, and D168N/A225V/K440N/3X) exhibited higher hydroxylation activity toward indole in comparison to parent enzyme. Moreover, using saturation site-directed mutagenesis at amino acid positions 168, 225, 434, 435, and 440, two P450 BM-3 variants (D168H/3X, E435T/3X) with an up to sixfold increase in catalytic efficiency (k _cat/K _m) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3-hydroxy-indole) compared to parent mutant (93 vs72%).     

A Compound I Mimic Reveals the Transient Active Species of a Cytochrome P450 Enzyme: Insight into the Stereoselectivity of P450-Catalysed Oxidations

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.     

支持向量机与KStar模型预测细胞色素P450酶催化的氧脱烃反应

分别以支持向量机(SVM)和KStar方法为基础, 构建了代谢产物的分子形状判别和代谢反应位点判别的嵌套预测模型. 分子形状判别模型是以272个分子为研究对象, 计算了包括分子拓扑、二维自相关、几何结构等在内的1280个分子描述符, 考查了支持向量机、决策树、贝叶斯网络、k最近邻这四种机器学习方法建立分类预测模型的准确性. 结果表明, 支持向量机优于其他方法, 此模型可用于预测分子能否被细胞色素P450酶催化发生氧脱烃反应. 代谢反应位点判别模型以538个氧脱烃反应代谢位点为研究对象, 计算了表征原子能量、价态、电荷等26个量子化学特征, 比较了决策树、贝叶斯网络、KStar、人工神经网络建模的准确率. 结果显示, KStar模型的准确率、敏感性、专一性均在90%以上, 对分子形状判别模型筛选出的分子, 此模型能较好地判断出哪个C―O键发生断裂. 本文以15个代谢反应明确的中药分子为验证集, 验证模型准确性, 研究结果表明基于SVM和KStar的嵌套预测模型具有一定的准确性, 有助于开展中药分子氧脱烃代谢产物的预测研究.     

Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

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.