【作者】 李锦莲；

【导师】 苏忠民；

【作者基本信息】 延边大学 ， 有机化学， 2011， 博士

【摘要】 DNA G-四链体因其能够抑制端粒酶的活性以及致癌基因的表达,已成为抗癌药物研究的新靶点。然而目前多数DNA G-四链体配体因对不同结构的G-四链体选择性差而阻碍了其临床应用。G-四链体结构沟槽的多态性,以及与B-DNA沟槽结构的差异性,使针对DNA G-四链体沟槽的药物设计成为人们研究的热点。然而由于实验手段的限制,对配体与DNA G-四链体沟槽作用机制还不清楚。分子模拟技术在研究药物与靶点之间相互作用机制方面,展示了实验不可比拟的优势。本论文中,主要采用分子力学、分子动力学、分子对接等模拟方法,在DNA G-四链体沟槽位点的结构,药物配体与不同G-四链体沟槽的作用机制等方面进行了研究,通过探讨配体与沟槽相对长度、沟槽宽度以及配体的构型等因素对配体在DNA G-四链体沟槽中作用的影响,为针对不同DNA G-四链体沟槽的药物设计提供理论依据。另外,本论文还研究了哌啶类拮抗剂对hCCR3/hCCR1受体的选择性作用机制,为选择性hCCR3受体拮抗剂的结构修饰和改造提供理论依据。本论文的主要内容包括：1、利用分子动力学、量子化学计算等手段研究了Dist-A二聚体在不同长度平行型DNA G-四链体沟槽中的作用机制,发现Dist-A二聚体在d[TGGGGT]4序列形成的G-四链体沟槽中,能够很好地契合在四链体沟槽的底部,并且Dist-A的酰胺基末端能够与沟槽3’末端鸟嘌呤碱基上的N3形成稳定的氢键；而在d[GGGGGG]4序列形成的更长的G-四链体鸟嘌呤沟槽中,Dist A二聚体明显移向5’末端,其中配体L2的酰胺基末端从沟槽中间移出。动力学模拟结果表明,Dist A二聚体与沟槽末端的鸟嘌呤碱基有更好的结合能力。为进一步探讨构成G-四链体沟槽的鸟嘌呤碱基的性质,利用密度泛函方法计算了三层连续G-四集体鸟嘌呤核心的自然电荷(NBO电荷),发现由于金属阳离子的存在,中间层06原子的负电荷比末端的06原子多0.05|e|,而面向沟槽的N3原子负电荷相应减少。表面静电势计算结果也表明,中间层G-四集体具有比末端更偏正的静电势。中心金属阳离子的存在对连续G-四集体鸟嘌呤核心的电子性质造成的这种影响,使沟槽末端对阳离子配体的结合更有利。这项研究提出了药物在平行型G-四链体沟槽中结合的影响因素,对基于平行型DNA G-四链体沟槽的药物设计具有重要的指导意义。2、利用分子对接、分子动力学等手段研究了Dist-A及其衍生物B与反平行型端粒DNA G-四链体的作用机制,结果表明Dist-A主要通过平面堆积方式作用在反平行型端粒DNA G-四链体的末端；而Dist-A衍生物B则由于更长的链状结构,使其通过平面堆积和沟槽结合共同作用的方式与反平行型端粒DNA G-四链体作用。MM GBSA计算进一步表明,Dist-A衍生物B在反平行型端粒DNA G-四链体宽沟槽位点的结合具有最好的稳定G-四链体作用。3、利用分子对接、分子动力学等手段研究了类固醇衍生物(Steroid FG)与杂化型端粒DNA G-四链体和B-DNA的作用机制,比较了Steroid FG在两种沟槽中作用的差异性。结果表明Steroid FG在杂化型端粒DNA G-四链体中处于沟槽和侧面loop共同形成的疏水区,以稠合四环上的两个甲基面向沟槽的方式,通过疏水和范德华作用结合；而在B-DNA中,配体的稠合四坏凹侧冲向沟槽底部的碱基,通过两端的氨基与DNA形成氢键,使配体稳定在TA小沟槽区。MM_PBSA计算表明SteroidFG在杂化型端粒DNA G-四链体中有更低的结合自由能。此项研究不仅探讨了结构特殊的类固醇类药物与端粒DNA G-四链体的相互作用机制,并且指出了其对端粒DNA G-四链体具有更好选择性的原因,对发展新型靶向端粒G-四链体沟槽配体具有积极的指导意义。4、利用配体支持同源模建(ligand-supported homology modeling)方法,以牛视网膜紫质X射线衍射晶体结构为模板,参考定位突变实验数据,模建了拮抗剂键合(antagonist-bound)的人类趋化因子受体hCCR3和(?)hCCR1的三维结构模型。并将一系列1,4-二取代哌啶类拮抗剂分别对接进精致的hCCR3和hCCR1受体模型中,以配体在hCCR3中的结合自由能理论值对-IgIC50值做线性回归,确定性系数R2为0.94,证明模型是合理的。分析对接结果发现配体主要通过疏水芳香作用与hCCR3和hCCR1受体结合,而4-苄基哌啶类拮抗剂对hCCR3受体产生选择性的主要原因在于配体的哌啶环与hCCR3和hCCR1受体中Tyr255 (TM7)的苯酚侧链不同的作用方式。而且hCCR3中处于结合口袋的EL2区的疏水性也为拮抗剂对hCCR3受体的选择性做出了贡献。这些结果可以为hCCR3受体选择性拮抗剂的发现及结构改造提供理论依据。更多还原

【Abstract】 Human DNA G-quadruplexes have been an attractive drug target for cancer therapeutic intervention. Especially the G-quadruplex groove has been paid growing attention to because of its selectivity. With the development of computer, molecular docking and molecular dynamics simulation have been applied in the study of the interaction mechanism between drug ligand and macromolecular and drug design. In this work, the mechanism of drug ligand targeting the groove of DNA G-quadruplex and the selectivity of 4-benzylpiperidine antagonists for hCCR3 were studied.1. In this work, molecular dynamics (MD) simulations of different sequences of G-quadruplexes ([d(TGGGGT)]4 and [d(GGGGGG)]4) complex with distamycin A (Dist-A) dimmers were performed. The characteristics of drug binding in G-quadruplex groove was investigated. The simulations revealed that in the [d(TGGGGT)]4 groove, Dist-A located in the floor of the groove and the N3 atom of guanine base in the 3’-end of the groove can form stable hydrogen bond with the formyl amino terminus of Dist-A. While in the groove of [d(GGGGGG)]4, Dist-A dimmer located in the 5’-end of the groove, and the formyl amino terminus of L2 is repulsed by the guanine base in the middle of the groove and forms hydrogen bond (L2NH1-4/A_G3:O2P) with the phosphate backbone of the opposite strand A. The MD results indicates that it is more favorable for the binding of Dist-A in the end of groove. The density functional theory (DFT) method at the B3LYP/6-31G (d, p) level was utilized to estimate electronic properties of guanine bases of three successive G-tetrads. It is suggested that N3 atoms of guanine bases in the middle G-tetrad layers have less negative charge than that in the terminal ones, which would have disadvantageous effect for the binding of the formyl amino terminus of Dist-A in the middle groove of G-quadruplex. This study proposes the factors affecting the binding of drugs in the G-quadruplex groove, and is of significance for drug design based on the structure of G-quadruplex groove.2. The binding mechanism of Dist-A and Dist-A derivative B in human telomeric DNA antiparallel G-quadruplex were studied by molecular docking and molecular dynamics (MD) simulation. MD simulation indicated that Dist-A canπ-stacking interact with the end of the quartet, while Dist-A derivative B interact with antiparallel G-quadruplex in L shape (stacking on the end of the quartet and binding in the groove). The analysis of the widths of different complexes showed that in the complexes of ligands binding in wide and meddle grooves, the interactions of ligands and the backbones of loops change the widths of the grooves, while in the complexes of ligands binding in narrow groove, the width of groove were changed by the interaction between ligand and groove. MM_PBSA calculation indicated that Dist-A derivative B can stabilize the antiparallel G-quadruplex more than Dist-A. The entropy is more favorable for groove binding.3. The mechanisms of Steroid FG interaction with mixed hybrid-type DNA G-quadruplex and B-DNA were studied using molecular docking and molecular dynamics (MD) simulation. The selectivity of Steroid FG for hybrid-type DNA G-quadruplex and B-DNA was discussed. The results showed that in the hybrid-type DNA G-quadruplex, Steroid FG bound in the region between groove and flank loop with the mode of the two methyls on the fused ring facing to the quartet, while in the B-DNA, Steroid FG bound in the TA minor groove with the flank of the fused ring for the floor of groove. MM_PBSA calculation indicated that the complex of Steroid FG and G-quadruplex had lower binding free energy and more favorable binding entropy. This work may provide useful principles for drug designing based on G-quadruplex groove.4. The "antagonist-bound" three-dimensional models of the hCCR3 and hCCRl receptors were constructed by homology modeling, using the X-ray structure of bovine rhodopsin as the template, and refined with MM/MD methods. We also carried out an automated docking of several 1,4-disubstituted piperidine antagonists into the hCCR3 and hCCRl model. The binding hypothesis and the reliability of the hCCR3 model were confirmed by the reasonable correlation between the estimated binding free energy and the experimental -1gIC50 values (R2=0.94). The docking results reveal that the face-to-face Vander Waals interaction between piperidine ring and Tyr255 plays an important role on the selectivity of 4-phenzylpiperidine antagonists to hCCR3. Simultaneously, the more hydrophobic of the EL2 in hCCR3 is favorable for the selectivity of 4-phenzylpiperidine antagonists. This work would be beneficial for the rational design of hCCR3 receptor antagonist.更多还原