基于分子对接的苯丙素甙（PPGs）类化合物的虚拟筛选和合理设计

采用虚拟化合物生成法对抗肿瘤的苯丙素甙(PPGs)类化合物进行了配体受体对 接研究。以三种不同的骨架结构为基础分别生成了五十个虚拟苯丙素甙(PPGs)类化 合物，并将它们与端粒DNA受体进行分子对接，分析已知结构的对接结果，通过虚 拟筛选的方法得到了一批与受体相互作用能较高并且复合物能量较低的新的有潜力 的活性化合物。该方法可以弥补分子对接研究中，只能计算药物与受体的相互作用 ，无法有效设计新化合物的不足。这种方法在基于结构的药物分子设计中具有重要 的意义。     

苏木素与DNA相互作用的光谱研究

以吖啶橙(AO)作探针研究了苏木素(HE)与DNA的相互作用. 吸收光谱和荧光光谱研究表明, 苏木素与DNA发生作用生成了复合物. 其结合比n_HE∶n_DNA＝3∶1, 22 ℃时苏木素与DNA的结合常数K＝5.96×10⁴ L/mol. 同时研究了酸度、盐效应和温度等对苏木素与DNA相互作用的影响以及它们之间的作用方式, 确定了苏木素与鲱鱼精DNA之间为混合作用方式.     

超分子体系中的分子识别研究23.多胺修饰β-环糊精及其铜配合物与萘衍生物的包结配位作用

用荧光光谱滴定法测定了单-[6-(二乙烯三胺)-6-脱氧]-β-环糊精(1)、单-[6-(三乙烯四胺)-6-脱氧]-β-环糊精(2)及其铜配合物(3,4)与一系列萘衍生物在磷酸缓冲溶液(pH 7.2,0.1 mol·dm-3)中,25℃时形成超分子体系的稳定常数,并与母体β-环糊精的配位能力进行了比较.化学计量法表明,四种化学修饰β-环糊精与萘衍生物形成了1:1的超分子配合物.从尺寸适合、几何互补及多点识别等方面讨论了主体化合物对模型底物的分子选择性键合能力.结果表明,疏水相互作用、范德华力、静电相互作用及氢键等多种非共价键弱相互作用协同贡献于超分子配合物的形成,主-客体间的结构匹配在分子受体选择性键合底物形成超分子配合物中起重要作用.     

同步荧光法研究大蒜辣素与不同类型 蛋白的相互作用

研究大蒜辣素在大鼠血液中的分布及与不同蛋白质的相互作用。采用高效液相色谱法(HPLC)测定大蒜辣素在大鼠血浆及血细胞中的分布情况;荧光及同步荧光光谱法研究大蒜辣素及其转化产物硫化氢与牛血红蛋白、牛人血清白蛋白的相互作用。HPLC测定结果显示大蒜辣素进入血液后部分与血浆蛋白结合,部分进入血细胞后浓度需达到1100μg·mL~(-1)时才可检测到游离的大蒜辣素,同时可检测到有硫化氢产生;荧光法研究结果显示,大蒜辣素不与牛血红蛋白发生作用,而硫化氢可与牛血红蛋白相互作用;大蒜辣素可与牛及人血清白蛋白相互作用。大蒜辣素与不同类型的蛋白作用不同,为大蒜辣素的成药性研究提供数据支持。     

吖啶橙为荧光探针研究芹菜素与DNA的相互作用

在pH值为7.40的Tris-HC1缓冲溶液中,采用吸收光谱法、荧光光谱法以及粘度法研究了芹菜素(Ap)与鲱鱼精DNA(fsDNA)的相互作用.研究表明,Ap与fsDNA相互作用生成了结合比nAp:nDNA=2:1的复合物,温度300 K和310 K的结合常数Kb分别为1.068×104 L·m01-1和1.137×1...     

超分子体系强的分子识别研究 23: 多胺修饰β-环糊精及其铜 配合物与萘衍生 物的包结配位作用

用荧光光谱滴定法测定了单-[6-(二乙烯三胺)-6-脱氧]-β-环糊精(1)、单-[6-(三乙烯四胺)-6-脱氧]-β-环糊精(2)及其铜配合物(3,4)与一系列萘衍生物在磷酸缓冲溶液(pH7.2,0.1mol.dm^-^3)中,25℃时形成超分子体系的稳定常数,并与母体β-环糊精的配位能力进行了比较。化学计量法表明,四种化学修饰β-环糊精与萘衍生物形成了1:1的超分子配合物。从尺寸适合、几何互补及多点识别等方面讨论了主体化合物对模型底物的分子选择性键合能力。结果表明,疏水相互作用、范德华力、静电相互作用及氢键等多种非共价键弱相互作用协同贡献于超分子配合物的形成,主-客体间的结构匹配在分子受体选择性键合底物形成超分子配合物中起重要作用。     

超分子体系强的分子识别研究 23: 多胺修饰β-环糊精及其铜 配合物与萘衍生 物的包结配位作用

用荧光光谱滴定法测定了单-[6-(二乙烯三胺)-6-脱氧]-β-环糊精(1)、单-[6-(三乙烯四胺)-6-脱氧]-β-环糊精(2)及其铜配合物(3,4)与一系列萘衍生物在磷酸缓冲溶液(pH7.2,0.1mol.dm^-^3)中,25℃时形成超分子体系的稳定常数,并与母体β-环糊精的配位能力进行了比较。化学计量法表明,四种化学修饰β-环糊精与萘衍生物形成了1:1的超分子配合物。从尺寸适合、几何互补及多点识别等方面讨论了主体化合物对模型底物的分子选择性键合能力。结果表明,疏水相互作用、范德华力、静电相互作用及氢键等多种非共价键弱相互作用协同贡献于超分子配合物的形成,主-客体间的结构匹配在分子受体选择性键合底物形成超分子配合物中起重要作用。     

不同取代羟基黄酮类化合物与血清白蛋白的相互作用分析

采用荧光光谱法研究了3种具有不同取代羟基的黄酮类化合物芹菜素、染料木素和高良姜素与牛血清白蛋白(BSA)之间的相互作用,测定了3种黄酮化合物与BSA的结合常数和结合位点,并分析了它们对BSA的荧光猝灭过程及其之间的相互作用类型,同时利用同步荧光和紫外吸收光谱探讨了它们对BSA构象的影响.结果表明,3种黄酮类化合物分子中的羟基数目以及位置对各化合物与BSA的作用有重要影响,导致水溶液中这3种黄酮类化合物与BSA发生相互作用的强弱不同,其作用力顺序为芹菜素>高良姜素>染料木素.     

荧光法研究三种黄酮小分子与溶菌酶的相互作用

用荧光光谱法研究了芹菜素(Quercetin)、木犀草素(Luteocin)、槲皮素(Quercetin)与溶菌酶的相互作用, 对比了C3'—OH和C3—OH取代对黄酮与溶菌酶作用的影响. 结果表明, C3'—OH可大大增强黄酮与溶菌酶之间的作用, C3—OH的取代则导致作用力减弱. 根据3种黄酮的结构参数, 初步分析了C3'—OH和C3—OH取代对黄酮与蛋白相互作用的影响.     

分子光谱法研究美他环素与人血清白蛋白的相互作用

用荧光、紫外等分子光谱法研究了美他环素(MC)与人血清白蛋白(HSA)的相互作用,考察了不同温度下MC与HSA的结合常数KA和结合位点数n,同时研究了Cu2+,Al3+,Pb2+,Ca2+和K+等金属离子对MC与HSA的结合性质的影响.基于F rster偶极-偶极非辐射能量转移机理确定了荧光给体HSA与受体MC间的结合距离.     

Snooker: a structure-based pharmacophore generation tool applied to class A GPCRs

G-protein coupled receptors (GPCRs) are important drug targets for various diseases and of major interest to pharmaceutical companies. The function of individual members of this protein family can be modulated by the binding of small molecules at the extracellular side of the structurally conserved transmembrane (TM) domain. Here, we present Snooker, a structure-based approach to generate pharmacophore hypotheses for compounds binding to this extracellular side of the TM domain. Snooker does not require knowledge of ligands, is therefore suitable for apo-proteins, and can be applied to all receptors of the GPCR protein family. The method comprises the construction of a homology model of the TM domains and prioritization of residues on the probability of being ligand binding. Subsequently, protein properties are converted to ligand space, and pharmacophore features are generated at positions where protein ligand interactions are likely. Using this semiautomated knowledge-driven bioinformatics approach we have created pharmacophore hypotheses for 15 different GPCRs from several different subfamilies. For the beta-2-adrenergic receptor we show that ligand poses predicted by Snooker pharmacophore hypotheses reproduce literature supported binding modes for ～75% of compounds fulfilling pharmacophore constraints. All 15 pharmacophore hypotheses represent interactions with essential residues for ligand binding as observed in mutagenesis experiments and compound selections based on these hypotheses are shown to be target specific. For 8 out of 15 targets enrichment factors above 10-fold are observed in the top 0.5% ranked compounds in a virtual screen. Additionally, prospectively predicted ligand binding poses in the human dopamine D3 receptor based on Snooker pharmacophores were ranked among the best models in the community wide GPCR dock 2010.     

Sensitive and Low-background Electrochemical Immunosensor Employing Glucose Dehydrogenase and 1,10-Phenanthroline-5,6-dione

We report a sensitive electrochemical immunosensor with a low electrochemical background level, which results from electrochemical-enzymatic (EN) redox cycling based on mediated electrochemical oxidation of an electro-inactive reductant (glucose) at 0.0 V. The EN redox cycling employs flavin adenine dinucleotide-dependent glucose dehydrogenase and 1,10-phenanthroline-5,6-dione (PD). When PD was compared with five common quinone-based electron mediators, PD enabled the highest signal-to-background ratio, due to a very low electrochemical background level. When EN redox cycling was applied to a sandwich-type immunosensor, parathyroid hormone (PTH) in serum could be detected with a very low detection limit of ∼0.1 pg/mL.     

Minimization of MC1R selectivity by modification of the core structure of alpha-MSH-ND

BACKGROUND: Melanocortin, through its distinct receptor subtypes, has many different effects. Receptor-selective ligands are required to reduce the undesirable effects of melanocortin. To investigate which conformation is preferable to a given melanocortin receptor subtype, a structural and functional analysis of the ligand-receptor interactions was made by studying the biological activity, the nuclear magnetic resonance structures, and the patterns of the ligand-receptor interaction for each receptor subtype by homology modeling analysis. RESULTS: Among the several analogues examined, [Gln(6)]alpha-melanocyte-stimulating hormone (MSH)-ND was found to have 10000 times less biological activity than alpha-MSH-ND for the MC1R, whereas, the potencies of both oligopeptides were comparable in both the melanocortin-3 receptor (MC3R) and MC4R. [Gln(6)]alpha-MSH-ND exhibited a type I' beta-turn that was similar to the type I beta-turn structure of alpha-MSH-ND. However, a remarkable structural difference was observed with respect to the side chain orientations of the sixth and seventh residues of [Gln(6)]alpha-MSH-ND, which were found to be mirror images of alpha-MSH-ND. By homology modeling analysis, the His(6) of alpha-MSH-ND was found to interact with the TM2 regions of all three receptors (Glu(94) of MC1R, Glu(94) of MC3R, and Glu(100) of MC4R), but [Gln(6)]alpha-MSH-ND did not. The phenyl ring of the D-Phe(7) residue of [Gln(6)]alpha-MSH-ND revealed an interaction with the TM3 regions of both the MC3R and MC4R (Ser(122) of MC3R or Ser(127) of MC4R). However, in the MC1R, these serine residues corresponded to Val(122), which contains two methyl groups that induce steric hindrance with D-Phe(7) of [Gln(6)]alpha-MSH-ND. This is a possible explanation for the biological activity of [Gln(6)]alpha-MSH-ND for the MC1R being significantly lower than that for either the MC3R or MC4R. CONCLUSIONS: Minimization of the MC1R selectivity whilst preserving its comparable potency for both the MC3R and MC4R could be achieved by modifying the D-Phe(7) orientation of alpha-MSH-ND, while maintaining the 'type I beta-turn'-like structure.     

GPCR structure-based virtual screening approach for CB2 antagonist search

The potential for therapeutic specificity in regulating diseases has made cannabinoid (CB) receptors one of the most important G-protein-coupled receptor (GPCR) targets in search for new drugs. Considering the lack of related 3D experimental structures, we have established a structure-based virtual screening protocol to search for CB2 bioactive antagonists based on the 3D CB2 homology structure model. However, the existing homology-predicted 3D models often deviate from the native structure and therefore may incorrectly bias the in silico design. To overcome this problem, we have developed a 3D testing database query algorithm to examine the constructed 3D CB2 receptor structure model as well as the predicted binding pocket. In the present study, an antagonist-bound CB2 receptor complex model was initially generated using flexible docking simulation and then further optimized by molecular dynamic and mechanical (MD/MM) calculations. The refined 3D structural model of the CB2-ligand complex was then inspected by exploring the interactions between the receptor and ligands in order to predict the potential CB2 binding pocket for its antagonist. The ligand-receptor complex model and the predicted antagonist binding pockets were further processed and validated by FlexX-Pharm docking against a testing compound database that contains known antagonists. Furthermore, a consensus scoring (CScore) function algorithm was established to rank the binding interaction modes of a ligand on the CB2 receptor. Our results indicated that the known antagonists seeded in the testing database can be distinguished from a significant amount of randomly chosen molecules. Our studies demonstrated that the established GPCR structure-based virtual screening approach provided a new strategy with a high potential for in silico identifying novel CB2 antagonist leads based on the homology-generated 3D CB2 structure model.     

Probing Polymer Chain Conformation and Fibril Formation of Peptide Conjugates

Covalent conjugates between a synthetic polymer and a peptide hormone were used to probe the molecular extension of these macromolecules and how the polymer modifies the fibril formation of the hormone. NMR spectroscopy of ¹⁵N labeled parathyroid hormone (PTH) was employed to visualize the conformation of the conjugated synthetic polymer, triggered by small temperature changes via its lower critical solution temperature. A shroud-like polymer conformation dominated the molecular architecture of the conjugated chimeras. PTH readily forms amyloid fibrils, which is probably the physiological storage form of the hormone. The polyacrylate based polymers stimulated the nucleation processes of the peptide.     

Molecular modeling of the human vasopressin V2 receptor/agonist complex

The V2 vasopressin renal receptor (V2R), which controls antidiuresis in mammals, is a member of the large family of heptahelical transmembrane (7TM) G protein-coupled receptors (GPCRs). Using the automated GPCR modeling facility available via Internet (http://expasy.hcuge.ch/swissmod/SWISS-MODEL.html) for construction of the 7TM domain in accord with the bovine rhodopsin (RD) footprint, and the SYBYL software for addition of the intra- and extracellular domains, the human V2R was modeled. The structure was further refined and its conformational variability tested by the use of a version of the Constrained Simulated Annealing (CSA) protocol developed in this laboratory. An inspection of the resulting structure reveals that the V2R (likewise any GPCR modeled this way) is much thicker and accordingly forms a more spacious TM cavity than most of the hitherto modeled GPCR constructs do, typically based on the structure of bacteriorhodopsin (BRD). Moreover, in this model the 7TM helices are arranged differently than they are in any BRD-based model. Thus, the topology and geometry of the TM cavity, potentially capable of receiving ligands, is in this model quite different than it is in the earlier models. In the subsequent step, two ligands, the native [arginine⁸]vasopressin (AVP) and the selective agonist [d-arginine⁸]vasopressin (DAVP) were inserted, each in two topologically non-equivalent ways, into the TM cavity and the resulting structures were equilibrated and their conformational variabilities tested using CSA as above. The best docking was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. Finally, the amino acid residues were indicated, mainly in TM helices 3-7, as potentially important in both AVP and DAVP docking. Among those Cys¹¹², Val¹¹⁵-Lys¹¹⁶, Gln¹¹⁹, Met¹²³ in helix 3; Glu¹⁷⁴ in helix 4; Val²⁰⁶, Ala²¹⁰, Val²¹³-Phe²¹⁴ in helix 5; Trp²⁸⁴, Phe²⁸⁷-Phe²⁸⁸, Gln²⁹¹ in helix 6; and Phe³⁰⁷, Leu³¹⁰, Ala³¹⁴ and Asn³¹⁷ in helix 7 appeared to be the most important ones. Many of these residues are invariant for either the GPCR superfamily or the neurophyseal (vasopressin V2R, V1aR and V1bR and oxytocin OR) subfamily of receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for the ligand affinity [Mouillac et al., J. Biol. Chem, 270 (1995) 25771].     

Using Homology Modeling, Molecular Dynamics and Molecular Docking Techniques to Identify Inhibitor Binding Regions of Somatostatin Receptor 1

The G protein coupled receptor(GPCR), one of the members in the superfamily, which consists of thousands of integral membrane proteins, exerts a wide variety of physiological functions and responses to a large portion of the drug targets. The 3D structure of somatostatin receptor 1(SSTR1) was modeled and refined by means of homology modeling and molecular dynamics simulation. This model was assessed by Verify-3D and Vadar, which confirmed the reliability of the refined model. The interaction between the inhibitor cysteamine, somatostatin(SST) and SSTR1 was investigated by a molecular docking program, Affinity. The binding module not only showed the crucial residues involved in the interaction, but also provided important information about the interaction between SSTR1 on the one hand and ligands on the other, which might be the significant evidence for the structure-based design.     

Investigation of calcium disorders.

The advent of new two-site immunometric assays for intact parathyroid hormone (PTH) measurement has enhanced the interpretation of results in many patients under investigation for hypercalcemia. The aim of this article is to give practical advice on the use of the new intact PTH methods and other tests for the investigation of the more common disorders of calcium metabolism.