氯沙坦对急性心房颤动心房肌L型钙离子通道的影响

目的探讨氯沙坦对家兔急性心房颤动模型的心房肌L型钙离子通道的影响。方法家兔30只随机分为0．9％氯化钠溶液起搏（NSP）组、氟沙坦起搏（LP）组。观测每组基础状态、快速心房起搏时心房有效不应期（AERP）及心房肌L-型钙通道的电流密度（ICa-L）进行统计学处理。结果快速起搏8h后NSP组AERP的下降较LP组显著,差异有统计学意义（P〈0．05）;LP组较NSP组心房肌ICa-L差异无统计学意义（P〉0．05）;但其标准差显著降低（P〈0．05）。结论氯沙坦可以抑制快速心房起搏引起AERP缩短及心房肌ICa-L离散度增加,从而减轻心房颤动（AF）的电重构。     

人类CCR5三维结构的同源模建

趋化因子受体(Chemokine receptor)是GPCR(G-protein coupled receptors)的超级家族成员,在各种免疫反应中有着重要的作用。CCR5是CC亚族趋化因子RANTES,MIP-1a,和MIP-1b的特异性受体。该文采用同源模建的方法,并通过胞外环区优化,动力学优化和能量最小化的方法初步得到了一个较为合理的CCR5的结构模型。说明该文采用的模建流程方法,在跨膜蛋白的同源模建中有着重要的作用,能给实际工作带来很好的指导作用。     

α-芋螺毒素特异性结合受体-神经型烟碱乙酰胆碱受体的同源模建及功能预测

目的探讨神经型烟碱乙酰胆碱受体能被其竞争性拮抗剂α-芋螺毒素选择性阻断的机理。方法采用同源模建的方法构建神经型烟碱乙酰胆碱受体α-7亚型的三维空间结构,并利用分子对接的方法与已知空间构像的α-芋螺毒素对接。结果已构建的神经型烟碱乙酰胆碱受体α-7亚型结构合理,并与已知空间构像的α-芋螺毒素对接成功。结论传统的烟碱型乙酰胆碱受体拮抗剂缺乏专一性,而靶向神经元神经型烟碱乙酰胆碱受体的α-芋螺毒素种类多,特异性强,与不同亚单位组成的受体亲和力不同,应用潜力很大。     

多巴胺D2受体三维结构预测及与激动剂配体相互作用的研究

以细菌视紫红质(bacteriorhodopsin)的三维晶体结构为模板,预测了多巴胺D2受体跨膜区的7个α螺旋肽段的三维结构。根据定点突变实验数据以及预测的受体三维结构,确定了激动剂配体结合腔由Asp86,Ser141,Ser144等12个氨基酸残基组成。为了校正和检验所得的模型,分别以一组刚性、一组柔性的激动剂与受体对接(DOCK),分析-logIC₅₀和结合能Eb相关性,较好的结果说明该模型是可靠的。     

戊乙奎醚光学异构体与毒蕈碱型受体亚型的分子对接研究

目的:研究戊乙奎醚光学异构体对毒蕈碱型(M)受体亚型的亲和性,为揭示戊乙奎醚的作用靶点及药效选择性提供参考。方法:利用同源建模和分子对接等分子模拟技术,通过比较戊乙奎醚不同光学异构体R1(3R,2′R)、R2(3R,2′S)、S1(3S,2′R)和S2(3S,2′S)与M受体亚型M_1~M_5的结合能,判断其与M受体亚型的亲和性。结果:戊乙奎醚4个光学异构体均能够对接到M受体各亚型的活性位点,不同异构体与不同M受体亚型的分子对接显示出较大差异。4个光学异构体与M_3受体具有较大的结合能,在5 736.519~5 907.143 kcal/mol之间;R1与M_1的结合能为1 190.04 kcal/mol;而其他光学异构体与各受体亚型的结合能较低或为负数。结论:戊乙奎醚的异构体R1对M_1受体具有亲和性,4个光学异构体均对M_3受体具有亲和性。     

eEF2K蛋白同源模建及其抑制剂小分子的虚拟筛选研究

目的:筛选潜在的真核生物延伸因子2激酶(eEF2K)抑制剂小分子,为eEF2K抑制剂的设计和研发提供参考。方法:采用同源模建技术构建eEF2K蛋白晶体结构模型,并进行Loop优化和分子动力学优化,借助SAVES在线服务器从Verify_3D、EERAT和拉氏图等3个方面对上述模型进行评估。收集55个eEF2K抑制剂小分子,使用InsightⅡ软件以其中的28个(编号为奇数,设为训练集)为基础构建具有活性预测能力的Hypogen药效团模型,以另外27个(编号为偶数,设为测试集)进行验证,通过拟合活性[即半数抑制浓度的负对数(p IC50)]预测值与真实值并借助Ligand profiler热图筛选最优药效团模型。结合上述药效团模型和Lipinski五规则、分子对接方法进行eEF2K抑制剂小分子的虚拟筛选。结果与结论:所建eEF2K蛋白晶体结构模型的整体质量因素得分为93.697,其中83.33%的氨基酸Verify_3D得分≥0.2,且位于不允许区的氨基酸占氨基酸总数的1.7%,其氨基酸构象及骨架结构合理,模型可靠性高。共构建了9个具有活性预测功能的Hypogen药效团模型(02～10号),其中03号药效团模型包含2个氢键受体和2个共轭芳香环,可更好地区分活性及非活性分子,其pIC_(50)预测值与真实值拟合最好(相关系数为0.665 3),具有较好的预测能力和较高的可靠性。通过虚拟筛选最终获得9个潜在的eEF2K抑制剂小分子(pIC_(50)预测值为1.074～1.185,分子与蛋白相互作用的Dcoking-score得分为-9.730～-7.467),其中Pro268、Asp267、Gln171、Phe121、Glu212可能是e EF2K抑制剂与靶点蛋白相互作用的关键氨基酸,作用方式包括氢键、盐桥、疏水等。上述分子有望成为e EF2K抑制剂研发的先导化合物。     

同源建模构建ABLSH3突变体及其与RIN1蛋白结合能力的分析

Bcr-Abl SH3与RIN1富含脯氨酸的序列结合,激活Bcr-Abl酪氨酸激酶活性,引起慢粒患者对伊马替尼耐药,为改变两者结合方式解决耐药问题,笔者对Bcr-Abl SH3结构域活性位点进行分析。Pep Site2.0软件分析RIN1与Bcr-Abl SH3结合部位及特点,选择突变位点,VMD1.8.7和NAMD2.6软件动态模拟获取稳定的SH3突变体结构,Pep Site2.0分析突变体和RIN1结合能力大小。两者结合部位位于SH3 90-118氨基酸位点,3种突变方式中双位点突变效果明显优于单位点及缩短的片段,单位点突变中并非所有被选择的位点突变后结合能力都提高,说明RIN1与SH3结合不仅依赖结合部位的特异氨基酸,其周围的氨基酸对维持两者的结合有着至关重要的作用。     

梭曼单链抗体酶EP_6一级结构的确定和三维结构的模建

以梭曼水解反应过渡态类似物 O1-甲基 - O2 -(1 ,2 ,2 -三甲基丙基 ) - 2 -羟基 - 5-硝基苯基甲基膦酸为半抗原得到了梭曼单链抗体酶 EP6.对这一株抗体进行了核苷酸序列的测定 ,测序结果表明这一单链抗体基因全长 71 4bp,其中重链长 351 bp,编码1 1 7个氨基酸 ,轻链长 31 8bp,编码 1 0 6个氨基酸 ,二者由编码 (Gly4 Ser) 3的 45bp的连接肽基因相连 .重 ,轻链均为开放读框 ,有明确的四个框架区和三个互补决定区 (CDR)以及抗体特征性的两个半胱氨酸残基 ,表明重 ,轻链的氨基酸序列符合鼠抗体可变区特征 .在 SGI工作站上对 EP6的三维结构进行了同源模建 .计算机模型显示 EP6表面包含一个由重轻链 CDRs围成的 ,可能用于半抗原结合和催化活性的长裂缝 ,其中轻链的 CDR3(L- CDR3)和重链三个 CDRs(H- CDRs)都可能参与抗原或半抗原的接触 ,L- CDR3和 H- CDR2的贡献较大 ,对抗体酶功能可能起重要作用     

梭曼单链抗体酶EP6一级结构的确定和三维结构的模建

以梭曼水解反应过渡态类似物O¹-甲基-O²-(1，2，2-三甲基丙基)-2-羟基-5-硝基苯基甲基膦酸为半抗原得到了梭曼单链抗体酶EP₆. 对这一株抗体进行了核苷酸序列的测定，测序结果表明这一单链抗体基因全长714 bp, 其中重链长351 bp，编码117个氨基酸，轻链长318 bp，编码106个氨基酸，二者由编码(Gly₄Ser)₃的45 bp的连接肽基因相连. 重, 轻链均为开放读框，有明确的四个框架区和三个互补决定区（CDR）以及抗体特征性的两个半胱氨酸残基，表明重，轻链的氨基酸序列符合鼠抗体可变区特征. 在SGI工作站上对EP₆的三维结构进行了同源模建. 计算机模型显示EP₆表面包含一个由重轻链CDRs围成的, 可能用于半抗原结合和催化活性的长裂缝，其中轻链的CDR3（L-CDR3）和重链三个CDRs(H-CDRs)都可能参与抗原或半抗原的接触，L-CDR3和H- CDR2的贡献较大，对抗体酶功能可能起重要作用.     

Regulation of Cav1.2 current: interaction with intracellular molecules

Ca(V)1.2 (alpha(1c)) is a pore-forming subunit of the voltage-dependent L-type calcium channel and is expressed in many tissues. The beta and alpha(2)/delta subunits are auxiliary subunits that affect the kinetics and the expression of Ca(V)1.2. In addition to the beta and alpha(2)/delta subunits, several molecules have been reported to be involved in the regulation of Ca(V)1.2 current. Calmodulin, CaBP1 (calcium-binding protein-1), CaMKII (calcium/calmodulin-dependent protein kinase II), AKAPs (A-kinase anchoring proteins), phosphatases, Caveolin-3, beta(2)-adrenergic receptor, PDZ domain proteins, sorcin, SNARE proteins, synaptotagmin, CSN5, RGK family, and AHNAK1 have all been reported to interact with Ca(V)1.2 and the beta subunit. This review focuses on the effect of these molecules on Ca(V)1.2 current.     

Optimization of Cav1.2 screening with an automated planar patch clamp platform

Introduction: Ca_v1.2 channels play an important role in shaping the cardiac action potential. Screening pharmaceutical compounds for Ca_v1.2 block is very important in developing drugs without cardiac liability. Ca_v1.2 screening has been traditionally done using fluorescence assays, but these assays have some limitations. Patch clamping is considered the gold standard for ion channel studies, but is very labor intensive. The purpose of this study was to develop a robust medium throughput Ca_v1.2 screening assay in PatchXpress^® 7000A by optimizing cell isolation conditions, recording solutions and experimental parameters. Under the conditions established, structurally different standard Ca_v1.2 antagonists and an agonist were tested. Methods: HEK-293 cells stably transfected with hCa_v1.2 L-type Ca channel were used. For experiments, cells were isolated using 0.05% Trypsin. Currents were recorded in the presence of 30 mM extracellular Ba²⁺ and low magnesium intracellular recording solution to minimize rundown. Ca_v1.2 currents were elicited from a holding potential of ? 60 mV at 0.05 Hz to increase pharmacological sensitivity and minimize rundown. Test compounds were applied at increasing concentrations for 5 min followed by a brief washout. Results: Averaged peak Ca_v1.2 current amplitudes were increased from 10 pA/pF to 15 pA/pF by shortening cell incubation and trypsin exposure time from 2.5 min at 37 °C to 1 min at room temperature and adding 0.2 mM cAMP to the intracellular solution. Rundown was minimized from 2%/min to 0.5%/min by reducing the intracellular free Mg²⁺ from 2.7 mM to 0.2 mM and adding 100 nM Ca²⁺. Under the established conditions, we tested 8 structurally different antagonists and an agonist. The IC₅₀ values obtained ranked well against published values and results obtained using traditional clamp experiments performed in parallel using the expressed cell line and native myocytes. Discussion: This assay can be used as a reliable pharmacological screening tool for Ca_v1.2 block to assess compounds for cardiac liability during lead optimization.     

Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Cav1.2 calcium channels

Background and purpose:

As a calcium channel blocker, diltiazem acts mainly on the voltage-gated calcium channels, Ca_v1.2, for its beneficial effects in cardiovascular diseases such as hypertension, angina and/or supraventricular arrhythmias. However, the effects of diltiazem on different isoforms of Ca_v1.2 channels expressed in heart and vascular smooth muscles remain to be investigated. Here, we characterized the effects of diltiazem on the splice variants of Ca_v1.2 channels, predominant in cardiac and vascular smooth muscles.

Experimental approach:

Cardiac and smooth muscle isoforms of Ca_v1.2 channels were expressed in human embryonic kidney cells and their electrophysiological properties were characterized using whole-cell patch-clamp techniques.

Key results:

Under closed-channel and use-dependent block (0.03 Hz), cardiac splice variant Ca_v1.2CM was less sensitive to diltiazem than two major smooth muscle splice variants, Ca_v1.2SM and Ca_v1.2b. Ca_v1.2CM has a more positive half-inactivation potential than the smooth muscle channels, and diltiazem shifted it less to negative potential. Additionally, the current decay was slower in Ca_v1.2CM channels. When we modified alternatively spliced exons of cardiac Ca_v1.2CM channels into smooth muscle exons, we found that all three loci contribute to the different diltiazem sensitivity between cardiac and smooth muscle splice isoforms.

Conclusions and implications:

Alternative splicing of Ca_v1.2 channels modifies diltiazem sensitivity in the heart and blood vessels. Gating properties altered by diltiazem are different in the three channels.     

Homology modeling of human CCR2 receptor

Homology model of CCR2 receptor was built on the basis of the crystal structure of human beta-2 adrenoceptor (PDB ID-2RH1). The model showed 99.3% residues in the core and allowed regions of the Ramachandran plot and there was no residue present in the disallowed regions. Prosa2003 program was used to assess the model and it displayed good native protein folding. The model also provided good root mean square deviation (RMSD) value and alignment score with the template, human β₂-adrenoceptor. The binding site is found within the transmembrane (TM) region and is sufficiently large enough for docking the known CCR2 ligands. The docking results validated the homology model to meet various criteria that are necessary in molecular modeling studies.     

Interactions of Calmodulin With the Multiple Binding Sites of Cav1.2 Ca2+ Channels

Although calmodulin binding to various sites of the Cav1.2 Ca²⁺ channel has been reported, the mechanism of the interaction is not fully understood. In this study we examined calmodulin binding to fragment channel peptides using a semi-quantitative pull-down assay. Calmodulin bound to the peptides with decreasing affinity order: IQ > preIQ > I-II loop > N-terminal peptide. A peptide containing both preIQ and IQ regions (Leu¹⁵⁹⁹ – Leu¹⁶⁶⁸) bound with approximately 2 mol of calmodulin per peptide. These results support the hypothesis that two molecules of calmodulin can simultaneously bind to the C-terminus of the Cav1.2 channel and modulate its facilitatory and inhibitory activities.     

Using property based sequence motifs and 3D modeling to determine structure and functional regions of proteins

Homology modeling has become an essential tool for studying proteins that are targets for medical drug design. This paper describes the approach we developed that combines sequence decomposition techniques with distance geometry algorithms for homology modeling to determine functionally important regions of proteins. We show here the application of these techniques to targets of medical interest chosen from those included in the CASP5 (Critical Assessment of Techniques for Protein Structure Prediction) competition, including the dihydroneopterin aldolase from Mycobacterium tuberculosis, RNase III of Thermobacteria maritima, and the NO-transporter nitrophorin from saliva of the bedbug Cimex lectularius. Physical chemical property (PCP) motifs, identified in aligned sequences with our MASIA program, can be used to select among different alignments returned by fold recognition servers. They can also be used to suggest functions for hypothetical proteins, as we illustrate for target T188. Once a suitable alignment has been made with the template, our modeling suite MPACK generates a series of possible models. The models can then be selected according to their match in areas known to be conserved in protein families. Alignments based on motifs can improve the structural matching of residues in the active site. The quality of the local structure of our 3D models near active sites or epitopes makes them useful aids for drug and vaccine design. Further, the PCP motif approach, when combined with a structural filter, can be a potent way to detect areas involved in activity and to suggest function for novel genome sequences.     

Modeling the 3D structure of GPCRs: advances and application to drug discovery

G protein-coupled receptors (GPCRs) are membrane-embedded proteins responsible for signal transduction; these receptors are, therefore, among the most important pharmaceutical drug targets. In the absence of X-ray structures, there have been numerous attempts to model the three-dimensional (3D) structure of GPCRs. In this review, the current status of GPCR modeling is evaluated, highlighting recent progress made in rhodopsin-based homology modeling and de novo modeling technology. Assessment of recent rhodopsin-based homology modeling studies indicates that, despite significant progress, these models do not yield hit rates that are sufficiently high for in silico screening (10 to 40% when screening for known binders). In contrast, the PREDICT modeling algorithm, which is independent of the rhodopsin structure, has now been fully validated in the context of drug discovery. PREDICT models are successfully used for drug discovery, yielding excellent hit rates (85 to 100% when screening for known binders), leading to the discovery of nanomolar-range new chemical entities for a variety of GPCR targets. Thus, 3D models of GPCRs should now allow the use of productive structure-based approaches for drug discovery.     

Homology modeling of G-protein-coupled receptors and implications in drug design

G-protein-coupled receptors (GPCRs) are considered therapeutically important due to their involvement in a variety of processes governing several cellular functions, and their tractability as drug targets. A large percentage of drugs on the market, and in development stages, target the super family of the GPCRs. The enormous interest in GPCR drug design is, however, limited by the scarcity of structural information. The only GPCR for which a three dimensional (3D) structure is reported is bovine rhodopsin and it belongs to class A of the GPCR family. As a result, there has been considerable interest in alternative techniques, for example, homology modeling of GPCRs, in order to derive useful three dimensional models of other proteins for use in structure-based drug design. However, homology modeling of GPCRs is not straightforward, and encounters several problems, owing to the availability of a single structural template, as well as the low degree of sequence homology between the template and target sequences. There are several key issues which need to be considered during every stage of GPCR homology modeling, in order to derive reasonable 3D models. Homology modeling of GPCRs has been utilized increasingly in the past few years and has been successful, not only in furthering the understanding of ligand-protein interactions, but also in the identification of new and potent ligands. Thus, with the lessons learned from past experiences and new developments, homology modeling in case of GPCRs can be harnessed for developing more reliable three dimensional models. This, in turn, will provide better tools to use in structure-based drug design leading to the identification of novel and potent GPCR ligands for several therapeutic indications.     

Homology modeling of rat and human cytochrome P450 2D (CYP2D) isoforms and computational rationalization of experimental ligand-binding specificities

The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1-4 and human CYP2D6, were investigated by measuring IC(50) values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with K(m) and V(max) values ranging between 78 and 145 microM and 0.048 and 1.122 min(-1), respectively. To rationalize observed differences in the experimentally determined IC(50) values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K. Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC(50) values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC(50) values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.     

新型三维氨基酸结构描述符的研究及其在多肽QSAR中的应用

目的建立一套新型的氨基酸残基结构描述符，用此描述符进行多肽的定量构效关系研究。方法通过理论计算得出167个氨基酸结构描述参数，再对167个参数进行主成分分析，从中提取出与氨基酸残基的三维结构性质密切相关的三个结构描述参数c₁，c₂，c₃-scales。然后采用G/PLS算法，建立氨基酸残基结构描述符c-scales与多肽生物活性之间的函数关系，确定多肽的QSAR模型。结果用此方法建立的苦味二肽、ACE抑制剂二肽和缓激肽增效剂五肽的QSAR模型，均有较好的拟合和预测能力，交叉验证相关系数和相关系数都在0.70以上。结论氨基酸残基结构描述符c-scales能定量的描述氨基酸的三维结构性质，而且利用此描述符能建立预测能力较强的多肽QSAR模型。