蛋白激酶CK2抑制剂作用机理的分子模拟研究及其靶向设计

发布时间：2018-02-22 09:59

本文关键词： 蛋白激酶CK2 分子模拟抑制剂三环喹啉类化合物香豆素　出处：《北京工业大学》2016年博士论文　论文类型：学位论文

【摘要】：蛋白激酶CK2是一种真核细胞中普遍存在的丝氨酸/苏氨酸蛋白激酶,与神经退行性病变、炎症和肿瘤等严重威胁人类健康的疾病密切相关,以其为靶点的抗肿瘤药物研发具有重要的临床应用价值和良好的应用前景。CK2全酶是由催化亚基α和调节亚基β构成的四聚体结构,对CK2激酶的生物学功能和结构稳定性至关重要。CK2抑制剂分为ATP竞争性抑制剂和阻断CK2α与CK2β亚基结合的抑制剂等。ATP竞争性抑制剂主要通过疏水和极性作用,定位于由疏水区、铰链区和正电区组成的激酶活性位点。环肽抑制剂作用于CK2α/CK2β结合位点,可有效干扰α与β亚基结合进而抑制CK2活性,已成为CK2抑制剂新的研究热点。本论文围绕CK2抑制剂开展了作用机制研究以及先导化合物的结构优化设计,为靶向CK2抑制剂的研发提供了理论依据和实验指导。ATP竞争性抑制剂三环喹啉类化合物已进入临床II期研究,可有效治疗多种实体肿瘤并展现了良好的药物代谢动力学特性。应用分子对接和三维定量构效关系和分子动力学模拟方法,针对一系列三环喹啉类化合物,构建了三维定量构效关系模型并阐释了此类化合物的结合模式。结果表明,三环喹啉类化合物的羧酸和吡啶取代基,分别与CK2α活性位点的正电区和铰链区形成极性作用,保证了此类化合物的高抑制活性。在化合物的骨架中,引入氢键供体基团和吸电子基团,可以增强化合物与铰链区和正电区的相互作用,提高化合物的CK2抑制活性。研究揭示了三环喹啉类化合物的作用模式及关键基团,同时基于CK2α活性位点结构,提出了针对此类化合物的结构优化修饰方案,为三环喹啉类化合物的结构设计提供了新的优化思路。为探究三环喹啉类化合物羧酸和吡啶基团的改变导致其抑制活性降低的结构机制,应用分子对接、分子动力学模拟和能量分析方法进行了系统深入的分析。结果表明,羧酸取代基变为非可电离基团或其位置发生改变时,化合物将无法与正电区残基形成稳定或合理的极性作用。而吡啶基团的氮原子变为氢原子或位置改变时,将导致化合物无法与铰链区残基形成氢键。两个关键基团的改变明显影响了化合物与CK2的结合模式,进而导致三环喹啉类化合物的CK2激酶抑制活性降低。化合物与CK2α结合模式的改变,还会诱导CK2α的柔性区域(C-loop,G-loop和β4/β5 loop)发生构象改变。研究证明了羧酸和吡啶基团是保证三环喹啉类化合物抑制活性的关键基团,揭示了该类化合物与CK2α之间无序的相互作用诱导CK2α柔性区域构象改变的结构机制,可为高活性CK2-ATP竞争性抑制剂的设计优化提供理论指导。7-羟基香豆素衍生物也是一类CK2-ATP竞争性抑制剂,且因具有较强抗肿瘤细胞增殖和抗氧化生物活性,成为重要的抗癌先导化合物。为提高此类化合物的抑制活性,基于不同类别ATP竞争性抑制剂与CK2的结合模式,针对7-羟基香豆素骨架结构,定向铰链区极性残基,设计优化并合成了氟代-7-羟基香豆素衍生物。生物活性评价结果表明,8-氯-7-羟基-4-三氟甲基香豆素具有较高的CK2抑制活性(IC50=0.4μM)和良好的抗肺癌细胞A549的增殖能力(IC50=20.15μM)。其抑制活性明显优于类似物8-氯-7-羟基-4-甲基香豆素(CK2抑制活性为IC50=2.2μM,A549抑制活性为IC50=29.26μM)。研究提出了针对7羟基香豆素类化合物的优化修饰方案,获得了兼具抗肿瘤细胞增殖能力和CK2激酶抑制活性的新型7-羟基香豆素衍生物,为以往单一作用模式CK2抑制剂的优化改造提供了一定的理论和实验指导。相对于传统ATP竞争性抑制剂而言,新型环肽抑制剂(Pc)可有效干扰α与β亚基的相互作用(IC50=3.0μM),从而间接抑制CK2对底物的磷酸化。环肽热点残基(Tyr188和Phe190)的突变会导致环肽抑制活性的明显降低(IC50100μM)。为了探究热点残基突变导致环肽抑制活性降低的结构机制,分别对野生型体系和两个突变复合物体系(Y188A和F190A)进行100纳秒长时分子动力学模拟,并结合主成分分析、动态相关性矩阵和自由能计算等方法对三个体系进行了深入的分析。结果表明,环肽与CK2α间形成了有序的疏水和极性相互作用网络,氢键主要维持环肽结构稳定以及环肽与CK2α的结合定位,而极性和疏水作用主要维持环肽残基与CK2α残基间的相互作用。两个突变体系中,有序的相互作用网络均遭到了不同程度的破坏,从而导致环肽抑制活性降低。在Y188A体系中,环肽与CK2α之间的关键氢键消失以及疏水作用减弱导致了环肽活性的降低。在F190A体系中,环肽结构稳定性的降低以及环肽与CK2α之间的重要疏水作用消失,使得环肽对CK2抑制活性显著降低。研究揭示了环肽关键残基突变导致CK2α与环肽亲和力下降的结构机制,证明了环肽与CK2α之间的有序疏水和极性相互作用网络是保证环肽抑制活性的关键,并成功识别了负责环肽结合与定位的关键作用残基,可为高活性干扰CK2α/CK2β相互作用抑制剂优化设计提供一定的指导。综上所述,论文采用多种分子模拟方法深入系统地探究了ATP竞争性抑制剂以及阻断CK2α/CK2β相互作用抑制剂的结合模式和作用机理,提出了抑制剂的结构优化修饰方案,获得了兼具抗肿瘤细胞增殖能力和CK2激酶抑制活性的新型7-羟基香豆素衍生物。上述研究为靶向CK2-ATP抑制剂先导化合物,以及CK2α/CK2β相互作用抑制剂的研发提供了理论支持和实验指导。
[Abstract]:Protein kinase CK2 is a ubiquitous eukaryotic serine / threonine protein kinase, and neurodegeneration, inflammation and tumor are closely related to a serious threat to human health, its antitumor drug target has important clinical application value and good application prospect of.CK2 enzyme is four the dimeric structure composed of a catalytic subunit alpha and beta subunit, biological function and structure stability of CK2 kinase inhibitor on.CK2 is divided into ATP competitive inhibitors and blocking CK2 alpha and CK2 beta subunit binding inhibitor.ATP competitive inhibitors mainly by hydrophobic and polar interactions, located in the hydrophobic area composition kinase active site hinge and positive region. Cyclic peptide inhibitors on CK2 alpha /CK2 beta binding sites, which can effectively interfere with alpha and beta subunits bind and inhibit the activity of CK2, has become the new CK2 inhibitors The focus of research. This thesis has carried out the mechanism research and structure optimization of lead compounds designed around CK2 inhibitors, CK2 inhibitors targeting research provides a theoretical basis and experimental guidance.ATP competitive inhibitors, tricyclic quinoline compounds have entered phase II clinical studies, to be effective in the treatment of various solid tumors and showed pharmacokinetic properties good. Application of molecular docking and QSAR and molecular dynamics simulation method for a series of tricyclic quinoline compounds, constructs a 3D QSAR model and explains the binding mode of this kind of compounds. The results show that tricyclic quinoline compound carboxylic acid and pyridine substituents, respectively, and the hinge region and positive region CK2 alpha active site polarity formation, ensure the high inhibitory activity of such compounds. The compounds of skeleton, introduced for hydrogen bonding The donor groups and electron withdrawing groups, can enhance the interaction of compounds with hinge and positive area, improve the compound CK2 inhibitory activity. The research reveals the role model and the key groups of tricyclic quinoline compounds, and based on CK2 alpha active site structure, puts forward the structure optimization for this kind of compounds modified scheme, provides optimization a new idea for the structural design of the tricyclic quinoline compounds. Cause the structure mechanism to reduce the inhibitory activity to explore the tricyclic quinoline compound carboxylic acid and pyridine group, application of molecular docking, the analysis of the system simulation and energy analysis method of molecular dynamics. The results showed that the carboxylic acid substituent into non ionizable groups the position is changed, will not be charged residues and compound zone formation polarity effect is stable or reasonable. And the nitrogen atom of the pyridine group into hydrogen Or change the position, will lead to compounds with the residues cannot form hydrogen bonds. The hinge region of two key groups changed significantly influence the binding mode of compounds with CK2, which led to the tricyclic quinoline compound CK2 kinase inhibitor activity. Compounds with CK2 alpha binding mode change, the flexible region will induce the CK2 alpha (C-loop, G-loop and beta 4/ beta 5 loop) conformational change. The study shows that carboxylic acid and pyridine group is the key group of inhibitory activity of tricyclic quinoline compounds, the structure mechanism reveals the interaction between the disordered compounds with CK2 alpha CK2 alpha induced changes in the conformation of the flexible region, for the high activity of CK2-ATP competition the optimization design of inhibitors provide theoretical guidance for.7- hydroxyl coumarin derivative is also a competitive inhibitor of CK2-ATP, and because of its strong anti - proliferation of tumor cells and antioxidant activity, as As an important anti-cancer lead compounds. In order to improve the inhibitory activity of these compounds, binding modes of different categories of competitive inhibitors of ATP and CK2 based on 7-, aiming at hydroxycoumarin skeleton structure, directional hinge region of polar residues, design optimization and synthesis of fluorinated -7- hydroxycoumarin derivatives. The biological activity evaluation results show that 8- chloro -7- three hydroxy -4- methyl coumarin fluoride with high CK2 inhibitory activity (IC50=0.4 M) and anti lung cancer cells A549 good proliferation ability (IC50=20.15 M). The inhibitory activity was significantly better than that of analogues of 8- chloro -7- hydroxy -4- methyl coumarin (CK2 IC50=2.2 M inhibitory activity, A549 inhibitory activity for IC50=29.26 M) study. Put forward the optimization scheme for the modification of 7 hydroxy coumarin compounds, new 7- hydroxy coumarin derivatives with anti tumor cell proliferation and inhibition of CK2 kinase activity was obtained, as the previous single Provides some theoretical and experimental guidance to optimize the transformation of the mode of action of CK2 inhibitors. Compared with the traditional ATP competitive inhibitors, new cyclic peptide inhibitors (Pc) interaction could effectively interfere with alpha and beta subunits (IC50=3.0, M), thereby indirectly inhibiting CK2 on substrate phosphorylation. The cyclic peptide hot spot residues (Tyr188 and Phe190) mutation may lead to a significant reduction in cyclic peptide inhibitory activity (IC50100 M). In order to explore the structure mechanism of hot spot residues mutations lead to cyclic peptide inhibitory activity decreased, respectively, of the wild type system and two mutant complexes (Y188A and F190A) by molecular dynamics simulation of 100 nanosecond long. Combined with principal component analysis, correlation matrix and dynamic free energy calculation method of three systems were analyzed. The results show that the cyclic peptide and CK2 alpha form orderly hydrophobic and polar interactions hydrogen bonding network, main dimension A ring of peptide structure stability and cyclic peptide with CK2 alpha binding position, while the polar and hydrophobic interactions mainly maintaining the interaction of cyclic peptide residues with CK2 alpha residues. Two mutations in the system, the interaction of network order were destroyed in different degrees, which leads to the decreased activity of cyclic peptide inhibition in Y188A system, between the cyclic peptide with CK2 alpha key hydrogen bonds and hydrophobic interactions weaken disappeared resulting in reduced cyclic peptide activity. In F190A system, disappear important hydrophobic interaction between structural stability and lower cyclic peptide cyclic peptide with CK2 alpha, the cyclic peptide inhibitory activity of CK2 decreased significantly. The research reveals the key residues of cyclic peptide mutations lead to decline of CK2 alpha and structure mechanism of cyclic peptide affinity, that the ordering between hydrophobic cyclic peptide with CK2 alpha and polar interaction network is the key to ensure the cyclic peptide inhibitory activity, and is responsible for the successful identification of cyclic peptide. The key role and location of residues, can interfere with CK2 alpha /CK2 beta interaction inhibitors provide some guidance for the optimization design of high activity. In summary, this paper uses a variety of molecular systematic studies of ATP competitive inhibitors and blocking the binding mode and action mechanism of CK2 alpha /CK2 beta inhibitors of the interaction simulation method, puts forward the structure optimization of inhibitors modification scheme, a novel 7- hydroxyl coumarin derivative has anti tumor cell proliferation and inhibition of CK2 kinase activity was obtained. The study of targeting CK2-ATP inhibitor lead compound, provides theoretical support and guidance and experimental research of CK2 alpha /CK2 beta interaction inhibitors.

【学位授予单位】：北京工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R91

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