Docking and molecular dynamics studies on CYP2D6

Drug-metabolizing enzymes, also known as cytochrome P450s, are a superfamily of hemoglobin responsible for metabolizing more than 90% clinical drugs. Cytochrome P450 2D6 (CYP2D6) is a significant member of cytochrome P450s for the reason of metabolizing about 20% clinical drugs. In this paper, molecular docking and molecular dynamic simulations are used to investi- gate the active site of CYP2D6, roles of essential amino acids within the active site and time-dependent protein energy changes. The results suggest that amino acids Glu216, Asp301, Ser304 and Ala305 in the active site are likely to form hydrogen bonding interac-tions with substrates; the benzene ring of Phe120 and aromatic ring in the substrates form ∏-∏ interactions. In addition, molecular dynamics simulations prove that the catalytic conformation of CYP2D6 without ligands can be obtained by their own atomic fluctuations. The impact of ligands on protein system energy and large conformational shift is not very large. Cytochrome P450s is known for their genetic polymorphisms, which will result in severe adverse drug reactions. Ideally, we hope to use mo- lecular modeling to investigate the differences between the substrates of wild-type and mutants while they are bonded with drugs, and predict the drug metabolizing ability of mutants. Reduce the possibility for people taking drugs that they can not metabolize, therefore reduce the rate of adverse drug reactions, and eventually establish a platform of personalized drugs to largely benefit human health.     

Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria.

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg+2 ions for the binding of some drugs. This structural analysis should facilitate rational drug design.     

EMND与人血清白蛋白的分子作用机制

微波条件下,运用一锅法合成了二氢嘧啶-2-酮的衍生物—5-乙酯基-6-甲基-4-(4-硝基苯基)-3,4-二氢嘧啶-2-酮(EMND).运用同步荧光光谱、位点竞争结合实验、荧光共振能量转移(FRET)理论以及分子模拟技术研究了EMND与人血清白蛋白(HSA)的分子作用机制.实验结果表明:EMND与HSA的结合位点位于色氨酸(Trp214)附近,EMND的结合位点位于HSA的IIA亚域.通过FRET理论计算得出EMND与色氨酸残基的结合距离r=4.25nm,说明EMND与HSA之间能够发生非辐射能量转移.分子模拟表明EMND结合到HSA IIA亚域的疏水空腔,它们之间存在氢键作用,进一步印证了同步荧光及位点竞争结合实验结果.本研究对于在分子水平上理解小分子与生物大分子的相互作用本质以及设计基于二氢嘧啶-2-酮的药物等都有一定的参考作用.     

The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism

Organisms encounter a wide range of foreign compounds--or 'xenobiotics'--with potentially harmful consequences. The cytochrome P450 (CYP) enzymes metabolize xenobiotics and thus are a primary defence against these compounds. Increased expression of specific CYP genes in response to particular xenobiotics is a central component of this defence, although such induction can also increase production of toxic metabolites. Here we show that the nuclear receptor CAR mediates the response evoked by a class of xenobiotics known as the 'phenobarbital-like inducers'. The strong activation of Cyp2b10 gene expression by phenobarbital, or by the more potent TCPOBOP, is absent in mice lacking the CAR gene. These animals also show decreased metabolism of the classic CYP substrate zoxazolamine and a complete loss of the liver hypertrophic and hyperplastic responses to these inducers. Cocaine causes acute hepatotoxicity in wild-type mice previously exposed to phenobarbital-like inducers and this toxicity is also absent in the CAR-deficient animals. Thus, loss of CAR function alters sensitivity to toxins, increasing or decreasing it depending on the compound. Modulation of CAR activity in humans may significantly affect metabolism of drugs and other xenobiotics.     

Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket

AcrB and its homologues are the principal multidrug transporters in Gram-negative bacteria and are important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel TolC and the membrane fusion protein AcrA. Minocycline and doxorubicin have been shown to bind to the phenylalanine cluster region of the binding monomer. Here we report the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin. These drugs bind to the access monomer, and the binding sites are located in the proximal multisite binding pocket, which is separated from the phenylalanine cluster region (distal pocket) by the Phe-617 loop. Our structures indicate that there are two discrete multisite binding pockets along the intramolecular channel. High-molecular-mass drugs first bind to the proximal pocket in the access state and are then forced into the distal pocket in the binding state by a peristaltic mechanism involving subdomain movements that include a shift of the Phe-617 loop. By contrast, low-molecular-mass drugs, such as minocycline and doxorubicin, travel through the proximal pocket without specific binding and immediately bind to the distal pocket. The presence of two discrete, high-volume multisite binding pockets contributes to the remarkably broad substrate recognition of AcrB.     

川丁特罗对大鼠细胞色素P450酶的影响

研究新型抗哮喘药川丁特罗（trantinterol）对大鼠细胞色素P450酶的影响. 大鼠连续7 d灌胃给予川丁特罗后, 测定肝微粒体中CYP450的质量摩
尔浓度和主要3种亚型CYP1A2,CYP2D6和CYP3A4活性的变化. 实验结果表明, 与对照组相比,  川丁特罗给药组的大鼠肝微粒体中CYP450的总质量摩尔浓度未受影响(P>0.05), 对主要亚型CYP1A2,CYP2D6和CYP3A4的活性也无影响(P>0.05), 表明该药物对肝微粒体中的主要代谢酶无抑制或诱导作用.     

Prediction of sites under adaptive evolution in flavin-containing monooxygenases: Selection pattern revisited

Flavin-containing monooxygenase (FMO), like cytochrome P450 (CYP), is a monooxygenase that uses the reducing equivalents of NADPH to reduce one atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. Recently, it was shown that some CYP isoforms have been subject to positive selection. However, it is unknown whether the highly conserved phase I detoxification enzyme, FMO, has undergone similar positive Darwinian selection. We used maximum-likelihood models of codon substitution, evolutionary fingerprinting, and cross species comparison to investigate the occurrence of adaptive evolution in FMO sequences. We used recent genomic data from a range of species, including vertebrates and invertebrates. We present the evidence for the occurrence of adaptive evolution in mammalian FMO 3, 4, 5, and fugu FMOs but not in mammalian FMO 1, FMO 2, frog FMOs, other fish FMOs and invertebrate FMOs. The sites under adaptive evolution were significantly associated with the insertion domain in mammalian FMO 5. We identified specific amino acid sites in FMOs 3–5 that are likely targets for selection based on the patterns of parallel amino acid change. The most likely role of adaptive evolution is the repair of mutations that permitted optimal NADP+ binding and improved catalytic efficiency. The occurrence of positive selection during the evolution of phase I detoxification enzymes such as FMOs 3–5 and fugu FMO suggests the occurrence of both high selection pressure acting on species within their unique habitats and significant changes in intensity and direction (forms of xenobiotics and drugs) resulting from changes in microhabitat and food.     

Structure of the human histamine H1 receptor complex with doxepin

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H(1) receptor (H(1)R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H(1)R complex with doxepin, a first-generation H(1)R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp?428(6.48), a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H(1)R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys?191(5.39) and/or Lys?179(ECL2), both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H(1)R antagonist specificity against H(1)R.     

Crystal structure of the channelrhodopsin light-gated cation channel

Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3?? resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.     

基于Scatchard法的碳酸酐酶-脂质体与药物结合常数测定

 为更好地模拟药物小分子与碳酸酐酶在体内的相互作用,采用碳酸酐酶-脂质体毛细管电泳法,以4-羧基苯磺酰胺为分析对象建立碳酸酐酶药物筛选模型,并以此模型为基础计算12 种药物小分子与碳酸酐酶的结合常数,分析药物小分子与碳酸酐酶的相互作用。结果表明,4-羧基苯磺酰胺与碳酸酐酶-脂质体的结合常数为1.172×10⁴ mL·g^-1,具有较强的相互作用。基于此,筛选出4 种(咖啡酸、L-抗坏血酸、2, 4-二氯-5-磺酰胺基苯甲酸和4-氯-3-磺酰胺基苯甲酸)和5种(阿魏酸、马兜铃酸、没食子酸、原儿茶酸、烟酸)与复合物具有较强或较弱相互作用的药物。通过该法可快速、有效、经济地测定碳酸酐酶或其他靶标与药物的相互作用,缩短药物研发周期。     

Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001

Cytochrome P450 17A1 (also known as CYP17A1 and cytochrome P450c17) catalyses the biosynthesis of androgens in humans. As prostate cancer cells proliferate in response to androgen steroids, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer, but drug development has been hampered by lack of information regarding the structure of CYP17A1. Here we report X-ray crystal structures of CYP17A1, which were obtained in the presence of either abiraterone, a first-in-class steroidal inhibitor recently approved by the US Food and Drug Administration for late-stage prostate cancer, or TOK-001, an inhibitor that is currently undergoing clinical trials. Both of these inhibitors bind the haem iron, forming a 60° angle above the haem plane and packing against the central I helix with the 3β-OH interacting with aspargine 202 in the F helix. Notably, this binding mode differs substantially from those that are predicted by homology models and from steroids in other cytochrome P450 enzymes with known structures, and some features of this binding mode are more similar to steroid receptors. Whereas the overall structure of CYP17A1 provides a rationale for understanding many mutations that are found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate a better understanding of the enzyme's dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers.     

Blast fungus-induction and developmental and tissue-specific expression of a rice P450<Emphasis Type="Italic">CYP72A</Emphasis> gene cluster

Cytochrome P450 gene superfamily is widely involved in diverse processes of plant development and environmental responses including defense response to pathogens.We previously isolated a rice cDNA fragment in a DD-PCR screening for blast fungus-induced genes. In the current study, we isolated a CYP72A gene cluster consisting of 7 P450 CYP72A genes (CYP72A17-23) with the conserved cDNA sequence through the public rice genome data. There are total 14 putative CYP72A members in the rice genome, with high diversity at N-terminal sequences while high homology at C-terminal sequences of those 14 putative proteins. We analyzed expression profiles of the cloned 7 CYP72A genes during pathogen infection and development. The results showed that expression of CYP72A18, 19, 22 and 23 was differentially regulated in the incompatible and compatible interactions between rice and blast fungus. Except CYP72A20, a pseudogene, other 6 CYP72A genes also exhibited temporal and spatial expression patterns, respectively.These findings provide fundamental data for rice P450 gene function analysis.     

Molecular dynamics simulations exploring drug resistance in HIV-1 proteases

Although HIV-1 subtype B still dominates the epidemic AIDS in developed countries, an increasing number of people in developing countries are suffering from an epidemic of non-subtype B viruses. What is worse, the efficacy of the combinational use of an-tiretroviral drugs is gradually compromised by the rapid development of drug resistance. To gain an insight into drug resistance, 10-ns MD simulations were simultaneously conducted on the complexes of the TL-3 inhibitor with 4 different proteases (B_wt, B_mut, F_wt and F_mut), among which the complex of the B_wt protease with the TL-3 inhibitor was treated as the control group. Detailed analyses of MD data indicated that the drug resistance of B_mut against TL-3 mainly derived from loss of an important hydrogen bond and that of F_wt was caused by the decrease of hydrophobic interactions in S1/S1’ pocket, while both of the two reasons mentioned above were the cause of the F_mut protease’s resistance. These results are in good agreement with the previous experiments, revealing a possible mechanism of drug resistance for the aforementioned protease subtypes against the TL-3 inhibitor. Additionally, another indication was obtained that the mutations of M36I, V82A and L90M may induce structural transforms so as to alter the inhibitor’s binding mode.     

凹土玉米芯垫料对大鼠肝微粒体细胞色素P450 酶系的影响

摘要: 目的观察凹土玉米芯垫料对大鼠肝脏细胞色素P450( CytP450) 、细胞色素b5( Cytb5) 含量及大鼠肝微粒 体CYP1A2 和CYP2E1 活性的影响。方法将SD 大鼠随机分为普通刨花组、玉米秸组、凹土玉米芯组和对照组,饲 养30 d、60 d、90 d 时测定大鼠肝脏微粒体CytP450、Cytb5 的含量和CYP1A2,CYP2E1 活性。结果60-90 d 时普 通刨花组、玉米秸组与空白对照组之间CytP450 含量有显著性差异( P ＜ 0. 05) ,凹土玉米芯组与对照组之间没有显 著性差异( P ＞ 0. 05) ; 肝微粒体Cytb5 含量、CYP1A2、CYP2E1 活性各组均没有明显差异( P ＞ 0. 05) 。结论凹土玉 米芯对大鼠肝脏细胞微粒体CytP450、Cytb5、CYP1A2 和CYP2E1 没有诱导或抑制作用,普通刨花组对大鼠肝 CytP450 有较强的诱导作用,玉米秸也有不同程度的诱导作用,但低于普通刨花的诱导作用。     

Structure of the C2 domain of human factor VIII at 1.5 A resolution

Human factor VIII is a plasma glycoprotein that has a critical role in blood coagulation. Factor VIII circulates as a complex with von Willebrand factor. After cleavage by thrombin, factor VIIIa associates with factor IXa at the surface of activated platelets or endothelial cells. This complex activates factor X (refs 6, 7), which in turn converts prothrombin to thrombin in the presence of factor Va (refs 8, 9). The carboxyl-terminal C2 domain of factor VIII contains sites that are essential for its binding to von Willebrand factor and to negatively charged phospholipid surfaces. Here we report the structure of human factor VIII C2 domain at 1.5 A resolution. The structure reveals a beta-sandwich core, from which two beta-turns and a loop display a group of solvent-exposed hydrophobic residues. Behind the hydrophobic surface lies a ring of positively charged residues. This motif suggests a mechanism for membrane binding involving both hydrophobic and electrostatic interactions. The structure explains, in part, mutations in the C2 region of factor VIII that lead to bleeding disorders in haemophilia A.     

Identification of the primary gene defect at the cytochrome P450 CYP2D locus

The mammalian cytochrome P450-dependent monooxygenase system is involved in the metabolism of drugs and chemical carcinogens. The role of these enzymes in toxicological response is exemplified by an autosomal recessive polymorphism at the cytochrome P450 CYP2D6 debrisoquine hydroxylase locus which results in the severely compromised metabolism of at least 25 drugs, and which in some cases can lead to life-threatening side-effects. In addition, this polymorphism, which affects 8-10% of the caucasian population, has been associated with altered susceptibility to lung and bladder cancer. Here we report the identification of the primary mutation responsible for this metabolic defect and the development of a simple DNA-based genetic assay to allow both the identification of most individuals at risk of drug side-effects and clarification of the conflicting reports on the association of this polymorphism with cancer susceptibility.     

Structural determinants for GoLoco-induced inhibition of nucleotide release by Galpha subunits

Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Galpha subunit by binding of GTP, the Galpha and Gbetagamma subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind to Galpha subunits and maintain G-protein subunit dissociation in the absence of Galpha activation. Here we describe the structural determinants of GoLoco activity as revealed by the crystal structure of Galpha(i1) GDP bound to the GoLoco region of the 'regulator of G-protein signalling' protein RGS14. Key contacts are described between the GoLoco motif and Galpha protein, including the extension of GoLoco's highly conserved Asp/Glu-Gln-Arg triad into the nucleotide-binding pocket of Galpha to make direct contact with the GDP alpha- and beta-phosphates. The structural organization of the GoLoco Galpha(i1) complex, when combined with supporting data from domain-swapping experiments, suggests that the Galpha all-helical domain and GoLoco-region carboxy-terminal residues control the specificity of GoLoco Galpha interactions.     

Structure of a beta1-adrenergic G-protein-coupled receptor

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.     

3个白桦细胞色素P450基因生物信息学及表达分析

【目的】研究白桦细胞色素P450基因表达的组织特异性,以及在茉莉酸甲酯(MeJA)、水杨酸(SA)、赤霉素(GA₃)、脱落酸(ABA)、乙烯利和伤害处理下的基因表达模式。【方法】筛选白桦转录组,获得3个BpCYP450 基因,分别命名为BpCYP4、BpCYP5、 BpCYP14,利用生物信息学分析BpCYP450蛋白的分子结构特征及其与其他物种CYP450蛋白的亲缘关系。采用QRT-PCR技术,对白桦BpCYP450组织特异性、激素信号及伤害诱导下的表达特征进行分析。【结果】生物信息学结果表明BpCYP4、BpCYP5、BpCYP14 cDNA序列长度分别为1 569、1 584和1 530 bp,具有完整的开放阅读框(ORF),分别编码522、527和509个氨基酸,均为亲水性跨膜蛋白,主要定位于叶绿体。白桦BpCYP450与百脉根、扁豆及豌豆等豆科植物CYP450蛋白亲缘性较高。组织特异性分析结果显示,3个BpCYP450 基因在叶和根中表达较高,茎中较低。激素信号及伤害诱导结果表明,3个BpCYP450基因不同程度地响应MeJA、SA、GA₃、ABA、乙烯利激素信号及伤害诱导。【结论】3个BpCYP450基因均为CYP450基因家族的新成员,具有组织特异性及激素诱导表达特性,可能在白桦生长发育、抵御胁迫及代谢物合成中发挥重要作用。