人类细胞色素P450 2E1酶在不同乙醇浓度下构象变化的分子动力学模拟（英文）

细胞色素P450(CYP)2E1家族酶是一种具有双重功能的单加氧酶,能够参与市场上6%药物的代谢而具有重要的作用.这类酶与酒精的消耗、糖尿病、肥胖症以及厌食症等密切相关,引起了广泛的研究兴趣.目前尚未见从原子水平上对这种酶在不同乙醇浓度下构象行为的研究.基于此,本文研究了花生四烯酸(AA)与CYP2E1复合物结构在不同乙醇浓度下构象与能量变化的特点.对于在不同乙醇浓度下AA与CYP2E1的复合物结构,采用分子动力学模拟结合自由能计算的方法进行研究.分子动力学模拟结果表明,His109和Lys243氨基酸残基对AA与CYP2E1的结合起到了至关重要的作用.当体系的乙醇浓度较高时,AA的结合能力有所下降,这种结合能力的下降是由于AA与CYP2E1之间氢键相互作用力的减弱所致.本研究对于AA与CYP2E1复合物结构在不同乙醇浓度下,AA分子与CYP2E1分子结合能力下降以及CYP2E1的构象变化给出了详细的解释.本研究工作得到的结论对于实验和理论研究均有重要意义,可为后续细胞色素P450酶类催化活性的研究提供理论支持.     

细胞色素p450 2s1(CYP2s1)三维结构模建及其与维甲酸分子的对接研究

利用同源模建和分子动力学模拟,模建了细胞色素P450(CYP2s1)的三维结构．在模建结构的基础上,分析了活性位点的组成和结构,并进行了与小分子(维甲酸)的分子对接研究．研究结果表明,在由维甲酸和CYP2s1形成的复合物中,非键相互作用较强,其中,GLu411和Ala414是与维甲酸相互作用能最强的两个残基,对复合物的结合起重要作用．     

细胞色素P450 2f1(CYP2f1)的三维结构模建和与维甲酸的对接研究

利用同源模建和分子动力学模拟方法，模建了细胞色素P450 2f1(CYP2f1)的三维结构．在此基础上，分析了活性位点的组成和结构，并进行了与小分子(维甲酸)的对接研究．研究结果表明，His328，Ser397和Arg417对复合物的结合起重要作用．     

UPLC-MS/MS结合多探针底物方法研究刺五加叶中黄酮苷类成分对CYP450活性的影响

利用超高效液相色谱-串联质谱联用(UPLC-MS/MS)的多反应监测(MRM)技术结合多探针底物方法, 研究了刺五加叶中的主要黄酮苷类化合物槲皮苷、金丝桃苷及芦丁对肝细胞色素P450酶(CYP450)亚型CYP1A2, CYP2C, CYP2E1, CYP2D和CYP3A活性的影响. 结果表明, 3种化合物对各CYP亚型酶均有抑制作用, 其中金丝桃苷和槲皮苷对CYP1A2催化的非那西丁的O-脱乙基反应抑制的IC₅₀值分别为46.53和49.75 μmol/L, 金丝桃苷和芦丁对CYP2E1催化的氯唑沙宗的6-羟基化反应抑制的IC₅₀值分别为99.87和86.36 μmol/L. 机理性抑制实验结果表明, 3种化合物对2种亚型酶的抑制作用是随着预孵时间延长而增强的机理性抑制.     

UPLC-TSQ-Endure质谱分析Rg_3对2型糖尿病大鼠P450酶活性的影响

应用超高效液相色谱-质谱联用法(UPLC-MS)结合多探针底物法建立了同时定量测定5个细胞色素P450酶探针底物的方法,同时考察正常大鼠和2型糖尿病大鼠CYP亚型酶活性的变化,分析人参皂苷Rg_3对CYP亚型酶的调控作用。结果表明,与正常组比较,2型糖尿病大鼠肝微粒体酶中P3A4和P2E1活性被诱导,P2D6,P1A2和P2C9活性被抑制。人参皂苷Rg_3诱导2型糖尿病大鼠P3A4和P1A2活性,抑制P2E1和P2C9活性,对P2D6活性无明显影响。人参皂苷Rg_3能够使糖尿病大鼠CYP2E1和CYP1A2亚型酶活性趋于恢复到正常生理状态,其在CYP450酶中的代谢产物为人参皂苷Rh2。方法适用于评价P450酶诱导或抑制的酶活性测定,在临床上指导2型糖尿病患者合理用药。     

CYP2C9酶与Warfarin结合模型的立体选择性理论研究

对CYP2C9酶与S-Warfarin复合物的晶体结构进行分子对接、分子动力学模拟、通道分析及结合自由能计算,发现原晶体结构中的结合模式为"亚稳态",提出了CYP2C9与S-Warfarin结合的可催化模式;比较了CYP2C9与S-和R-Warfarin结合的异同,确定了在结合过程中起重要作用的锚定氨基酸残基,尤其是位于活性位点区域的苯丙氨酸簇.在结合过程中这些残基通过芳香环的移动对稳定底物的结合模式起到至关重要的作用,阐明了该酶呈现相关底物选择性的原因.对于CYP2C9与底物对接模式及立体选择性的研究有助于在分子层面上理解特异性底物与酶的结合特点,为潜在的药物设计提供了合理可信的理论依据.     

基于多光谱方法和计算模拟的细胞色素CYP1A2与阿奇霉素的结合机制研究

为了解阿奇霉素(AZI)在模拟的人体环境中与CYP1A2的结合情况,探索AZI在人体内的转化机制,本论文使用分子对接、分子动力学模拟、荧光光谱、紫外分光光度法、傅立叶变换红外光谱、圆二色光谱等多种实验方法,阐明AZI与CYP1A2之间的相互作用机制。分子对接结果表明AZI与CYP1A2的结合方式是半包裹并且以疏水作用力相结合。分子动力学模拟结果表明,CYP1A2-AZI复合物的均方根偏差(RMSD)值增大;均方根波动(RMSF)值表明复合物体系柔性较大;回旋半径(Rg)值表明蛋白质特定区域的结构松散。荧光猝灭光谱实验表明,CYP1A2与AZI是以静态猝灭机制结合在一起。热力学参数表明AZI与CYP1A2之间的主导作用力为疏水作用力。时间分辨光谱表明AZI对CYP1A2的机制为静态猝灭。紫外光谱实验表明AZI与CYP1A2反应生成了复合物。红外光谱实验结果表明CYP1A2的二级结构含量发生了改变。圆二色光谱证实AZI对CYP1A2的二级结构产生影响。     

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.     

非经典三铂核药物与DNA作用的理论研究

利用分子力学、分子动力学和量子化学等计算方法研究了新型临床二期抗癌药物BBR3464([{trans-PtCl(NH3)2}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}]4+)与寡聚DNA片段复合物的几何构型及其电子结构. 结果表明, 利用分子力学和分子动力学确定的复合物结构与实验的基本吻合. BBR3464为+4价高电荷铂药, 与两端的铂相连的两个Cl配体间的距离是2.74 nm, 这使得药物与DNA交联速度快, 形成远程的1,4-链间交联. 计算结果表明, BBR3464与DNA识别能力强, 结合稳定. 所形成的复合物中既有Pt-N7间较强的配位键, 也存在许多氢键、弱氢键及静电作用. 复合物中结合位点及结合位点外的嘌呤碱基的构象发生了不同程度的改变. 复合物结构特征说明, DNA在键合药物后其构型并未发生定域的链弯曲, 而是离域的嘌呤碱基的构象转化, 其对DNA所造成离域性损伤与经典的药物不同. DNA是铂抗肿瘤药物的靶点, 多点键合和离域性损伤的结构特征与BBR3464的独特生物活性和临床表现相关.     

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。     

How Mutations Affecting the Ligand-receptor Interactions:a Combined MD and QM/MM Calculation on CYP2E1 and Its Two Mutants

Cytochrome P450(CYP) 2E1 is a dual function monoxygenase with a crucial role in the metabolism of 6% of drugs on the market at present. The enzyme is of tremendous interest for its association with alcohol consumption, diabetes, obesity and fasting. Despite the abundant experimental mutagenesis data, the molecular origin and the structural motifs for the enzymatic activity deficiencies have not been rationalized at the atomic level. In this regard, we have investigated the effects of mutation on the structural and energetic characteristics upon single point mutations in CYP2E1, N219D and S366C. The molecular dynamics(MD) simulation combined with quantum mechanics/molecular mechanics(QM/MM) and noncovalent interaction(NCI) analysis was carried out on CYP2E1 and its two mutants. The results highlight the critical role of Phe207, which is responsible for both structural flexibility and energetic variation, shortening the gap between the theory and the experimentally observed results of enzymatic activity decrease. The underlying molecular mechanism of the enzymatic activity deficiencies for mutants may be attributed to the changes of spatial position of Phe207 in the two mutants. This work provides particular explanations to how mutations affect ligand-receptor interactions based on combined MD and QM/MM calculations. Furthermore, the mutational effects on the activity of CYP2E1 obtained in the present study are beneficial to both the experimental and the computational works of CYPs and may allow researchers to achieve desirable changes in enzymatic activity.     

Molecular Docking of 3-Methylindole-containing Drugs Binding into CYP3A4

Drugs SPD-304(6,7-dimethyl-3-{[methyl-(2-{methyl-[1-(3-trifluoromethyl-phenyl)-1H-indol-3-ylmethyl]- amino}-ethyl)-amino]-methyl}-chromen-4-one) and zafirlukast contain a common structural element of 3-substituted indole moiety which closely relates to a dehydrogenated reaction catalyzed by cytochrome P450s(CYPs). It was reported that the dehydrogenation can produce a reactive electrophilic intermediate which cause toxicities and inactivate CYPs. Drug L-745,870(3-{[4-(4-chlorophenyl)piperazin-1-yl]-methyl}-1H-pyrrolo- 2,3-β-pyridine) might have similar effect since it contains the same structural element. We used molecular docking approach combined with molecular dynamics(MD) simulation to model three-dimensional(3D) complex structures of SPD-304, zafirlukast and L-745,870 into CYP3A4, respectively. The results show that these three drugs can stably bind into the active site and the 3-methylene carbons of the drugs keep a reasonable reactive distance from the heme iron. The complex structure of SPD-304-CYP3A4 is in agreement with experimental data. For zafirlukast, the calculation results indicate that 3-methylene carbon might be the dehydrogenation reaction site. Docking model of L-745,870-CYP3A4 shows a potential possibility of L-745,870 dehydrogenated by CYP3A4 at 3-methylene carbon which is in agreement with experiment in vivo. In addition, residues in the phenylalanine cluster as well as S119 and R212 play a critical role in the ligands binding based on our calculations. The docking models could provide some clues to understand the metabolic mechanism of the drugs by CYP3A4.     

CYP450 Epoxygenase Metabolites,Epoxyeicosatrienoic Acids,as Novel Anti-Inflammatory Mediators

Inflammation plays a crucial role in the initiation and development of a wide range of systemic illnesses. Epoxyeicosatrienoic acids (EETs) are derived from arachidonic acid (AA) metabolized by CYP450 epoxygenase (CYP450) and are subsequently hydrolyzed by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs), which are merely biologically active. EETs possess a wide range of established protective effects on many systems of which anti-inflammatory actions have gained great interest. EETs attenuate vascular inflammation and remodeling by inhibiting activation of endothelial cells and reducing cross-talk between inflammatory cells and blood vessels. EETs also process direct and indirect anti-inflammatory properties in the myocardium and therefore alleviate inflammatory cardiomyopathy and cardiac remodeling. Moreover, emerging studies show the substantial roles of EETs in relieving inflammation under other pathophysiological environments, such as diabetes, sepsis, lung injuries, neurodegenerative disease, hepatic diseases, kidney injury, and arthritis. Furthermore, pharmacological manipulations of the AA-CYP450-EETs-sEH pathway have demonstrated a contribution to the alleviation of numerous inflammatory diseases, which highlight a therapeutic potential of drugs targeting this pathway. This review summarizes the progress of AA-CYP450-EETs-sEH pathway in regulation of inflammation under different pathological conditions and discusses the existing challenges and future direction of this research field.     

Ligand Binding and Release Investigated by Contact-Guided Iterative Multiple Independent Molecular Dynamics Simulations

Binding and releasing ligands are critical for the biological functions of many proteins, so it is important to determine these highly dynamic processes. Although there are experimental techniques to determine the structure of a protein-ligand complex, it only provides a static picture of the system. With the rapid increase of computing power and improved algorithms, molecular dynamics (MD) simulations have diverse of superiority in probing the binding and release process. However, it remains a great challenge to overcome the time and length scales when the system becomes large. This work presents an enhanced sampling tool for ligand binding and release, which is based on iterative multiple independent MD simulations guided by contacts formed between the ligand and the protein. From the simulation results on adenylate kinase, we observe the process of ligand binding and release while the conventional MD simulations at the same time scale cannot.     

Theoretical studies on the conformational change of adenosine kinase induced by inhibitors

Adenosine kinase (AK) is a two‐domain protein that catalyzes the phosphorylation of adenosine to adenosine monophosphate. Inhibitors of AK could increase adenosine to levels that activate nearby adenosine receptors and produce a wide variety of therapeutically beneficial activities. To get insight into the interaction mechanism between inhibitors and AK, we chose two kinds of novel inhibitors, alkynylpyrimidine inhibitor (APy) and aryl‐nucleoside inhibitor (AN), and used docking and molecular dynamics simulation methods to study the conformational changes of human AK on binding inhibitors. The calculation results revealed that both APy and AN could induce conformational changes of AK and stabilize AK at different semiopen conformations. On binding APy, the small lid‐domain rotated 14°, and the binding pocket rearranged after MD simulation. But in AK‐AN complex, the rotation of small domain is 22°, and the sugar ring of AN is mobile in the binding pocket. Further docking calculations on APy analogues indicate that the semiopen conformation could well explain the SAR of AK inhibitors. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Molecular dynamics simulation and thermodynamic modeling of the self-assembly of the triterpenoids asiatic acid and madecassic acid in aqueous solution

The self-assembly behavior of the triterpenoids asiatic acid (AA) and madecassic acid (MA), both widely studied bioactive phytochemicals that are similar in structure to bile salts, were investigated in aqueous solution through atomistic-level molecular dynamics (MD) simulation. AA and MA molecules initially distributed randomly in solution were observed to aggregate into micelles during 75 ns of MD simulation. A "hydrophobic contact criterion" was developed to identify micellar aggregates from the computer simulation results. From the computer simulation data, the aggregation number of AA and MA micelles, the monomer concentration, the principal moments of the micelle radius of gyration tensor, the one-dimensional growth exhibited by AA and MA micelles as the aggregation number increases, the level of internal ordering within AA and MA micelles (quantified using two different orientational order parameters), the local environment of atoms within AA and MA in the micellar environment, and the total, hydrophilic, and hydrophobic solvent accessible surface areas of the AA and MA micelles were each evaluated. The MD simulations conducted provide insights into the self-assembly behavior of structurally complex, nontraditional surfactants in aqueous solution. Motivated by the high computational cost required to obtain an accurate estimate of the critical micelle concentrations (CMCs) of AA and MA from evaluation of the average monomer concentration present in the AA and MA simulation cells, a modified computer simulation/molecular-thermodynamic model (referred to as the MCS-MT model) was formulated to quantify the free-energy change associated with optimal AA and MA micelle formation in order to predict the CMCs of AA and MA. The predicted CMC of AA was found to be 59 microM, compared with the experimentally measured CMC of 17 microM, and the predicted CMC of MA was found to be 96 microM, compared with the experimentally measured CMC of 62 microM. The AA and MA CMCs predicted using the MCS-MT model are much more accurate than the CMCs inferred from the monomer concentrations of AA and MA present in the simulation cells after micelle self-assembly (2390 microM and 11,300 microM, respectively). The theoretical modeling results obtained for AA and MA indicate that, by combining computer simulation inputs with molecular-thermodynamic models of surfactant self-assembly, reasonably accurate estimates of surfactant CMCs can be obtained with a fraction of the computational expense that would be required by using computer simulations alone.     

Molecular dynamics study on the interaction of a mithramycin dimer with a decanucleotide duplex

The complex of a minor groove binding drug mithramycin (MTR) and the self-complementary d(TAGCTAGCTA) 10-mer duplex was investigated by molecular dynamics (MD) simulations using the AMBER 7.0 suite of programs. There is one disaccharide and trisaccharide segment projecting from opposite ends of an aglycone chromophore of MTR. A MTR dimer complex (MTR)2Mg2+ is formed in the presence of a coordinated ion Mg2+. A NMR solution structure of two (MTR)2Mg2+ complexes bound with one DNA duplex, namely, the 2:1 duplex complex, was taken as the starting structure for the MD simulation. The partial charge on each atom was calculated using the multiple-RESP fitting procedure, and all of the missing parameters in the Parm99 force field used were adapted comparably from the literature. The length of the MD simulation was 5 ns, and the binding free energy for the formation of a 1:1 or 2:1 duplex complex was determined from the last 4 ns of the simulation. The binding free energies were decomposed to components of the contributions from different energy types, and the changes in the helical parameters of the bound DNA duplex plus the glycosidic linkages between sugar residues of the bound MTR dimer were determined. It was found that binding of the first (MTR)2Mg2+ complex with the DNA duplex to form a 1:1 duplex complex does not cause stiffening of the duplex especially in the unoccupied site of the duplex. However, the overall flexibility of the DNA duplex is reduced substantially once the second (MTR)2Mg2+ complex is bound with the unoccupied site to form the 2:1 duplex complex. The van der Waals interactions were found to be dominant in the central part of the DNA duplex where sugar residues from each bound (MTR)2Mg2+ complex were inwardly pointing and the corresponding minor groove was widened.     

Exploring a model of a chemokine receptor/ligand complex in an explicit membrane environment by molecular dynamics simulation: the human CCR1 receptor

The seven transmembrane helices G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of signaling proteins found in humans. Homology modeling, molecular docking, and molecular dynamics (MD) simulation were carried out to construct a reliable model for CCR1 as one of the GPCRs and to explore the structural features and the binding mechanism of BX471 as one of the most potent CCR1 inhibitors. In this study, BX471 has been docked into the active site of the CCR1 protein. After docking, one 20 ns MD simulation was performed on the CCR1-ligand complex to explore effects of the presence of lipid membrane in the vicinity of the CCR1-ligand complex. At the end of the MD simulation, a change in the position and orientation of the ligand in the binding site was observed. This important observation indicated that the application of MD simulation after docking of ligands is useful. Explorative runs of molecular dynamics simulation on the receptor-ligand complex revealed that except for Phe85, Phe112, Tyr113, and Ile259, the rest of the residues in the active site determined by docking are changed. The results obtained are in good agreement with most of the experimental data reported by others. Our results show that molecular modeling and rational drug design for chemokine targets is a possible approach.     

Evaluation of the effect of the chiral centers of Taxol on binding to β-tubulin: A docking and molecular dynamics simulation study

Taxol is one of the most important anti-cancer drugs. The interaction between different variants of Taxol, by altering one of its chiral centers at a time, with β-tubulin protein has been investigated. To achieve such goal, docking and molecular dynamics (MD) simulation studies have been performed. In docking studies, the preferred conformers have been selected to further study by MD method based on the binding energies reported by the AutoDock program. The best result of docking study which shows the highest affinity between ligand and protein has been used as the starting point of the MD simulations. All of the complexes have shown acceptable stability during the simulation process, based on the RMSDs of the backbone of the protein structure. Finally, MM-GBSA calculations have been carried out to select the best ligand, considering the binding energy criteria. The results predict that two of the structures have better affinity toward the mentioned protein, in comparison with Taxol. Three of the structures have affinity similar to that of the Taxol toward the β-tubulin.