本文關(guān)鍵詞： 蛋白激酶CK2 分子模擬 抑制劑 三環(huán)喹啉類化合物 香豆素　出處：《北京工業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：蛋白激酶CK2是一種真核細胞中普遍存在的絲氨酸/蘇氨酸蛋白激酶,與神經(jīng)退行性病變、炎癥和腫瘤等嚴(yán)重威脅人類健康的疾病密切相關(guān),以其為靶點的抗腫瘤藥物研發(fā)具有重要的臨床應(yīng)用價值和良好的應(yīng)用前景。CK2全酶是由催化亞基α和調(diào)節(jié)亞基β構(gòu)成的四聚體結(jié)構(gòu),對CK2激酶的生物學(xué)功能和結(jié)構(gòu)穩(wěn)定性至關(guān)重要。CK2抑制劑分為ATP競爭性抑制劑和阻斷CK2α與CK2β亞基結(jié)合的抑制劑等。ATP競爭性抑制劑主要通過疏水和極性作用,定位于由疏水區(qū)、鉸鏈區(qū)和正電區(qū)組成的激酶活性位點。環(huán)肽抑制劑作用于CK2α/CK2β結(jié)合位點,可有效干擾α與β亞基結(jié)合進而抑制CK2活性,已成為CK2抑制劑新的研究熱點。本論文圍繞CK2抑制劑開展了作用機制研究以及先導(dǎo)化合物的結(jié)構(gòu)優(yōu)化設(shè)計,為靶向CK2抑制劑的研發(fā)提供了理論依據(jù)和實驗指導(dǎo)。ATP競爭性抑制劑三環(huán)喹啉類化合物已進入臨床II期研究,可有效治療多種實體腫瘤并展現(xiàn)了良好的藥物代謝動力學(xué)特性。應(yīng)用分子對接和三維定量構(gòu)效關(guān)系和分子動力學(xué)模擬方法,針對一系列三環(huán)喹啉類化合物,構(gòu)建了三維定量構(gòu)效關(guān)系模型并闡釋了此類化合物的結(jié)合模式。結(jié)果表明,三環(huán)喹啉類化合物的羧酸和吡啶取代基,分別與CK2α活性位點的正電區(qū)和鉸鏈區(qū)形成極性作用,保證了此類化合物的高抑制活性。在化合物的骨架中,引入氫鍵供體基團和吸電子基團,可以增強化合物與鉸鏈區(qū)和正電區(qū)的相互作用,提高化合物的CK2抑制活性。研究揭示了三環(huán)喹啉類化合物的作用模式及關(guān)鍵基團,同時基于CK2α活性位點結(jié)構(gòu),提出了針對此類化合物的結(jié)構(gòu)優(yōu)化修飾方案,為三環(huán)喹啉類化合物的結(jié)構(gòu)設(shè)計提供了新的優(yōu)化思路。為探究三環(huán)喹啉類化合物羧酸和吡啶基團的改變導(dǎo)致其抑制活性降低的結(jié)構(gòu)機制,應(yīng)用分子對接、分子動力學(xué)模擬和能量分析方法進行了系統(tǒng)深入的分析。結(jié)果表明,羧酸取代基變?yōu)榉强呻婋x基團或其位置發(fā)生改變時,化合物將無法與正電區(qū)殘基形成穩(wěn)定或合理的極性作用。而吡啶基團的氮原子變?yōu)闅湓踊蛭恢酶淖儠r,將導(dǎo)致化合物無法與鉸鏈區(qū)殘基形成氫鍵。兩個關(guān)鍵基團的改變明顯影響了化合物與CK2的結(jié)合模式,進而導(dǎo)致三環(huán)喹啉類化合物的CK2激酶抑制活性降低�；衔锱cCK2α結(jié)合模式的改變,還會誘導(dǎo)CK2α的柔性區(qū)域(C-loop,G-loop和β4/β5 loop)發(fā)生構(gòu)象改變。研究證明了羧酸和吡啶基團是保證三環(huán)喹啉類化合物抑制活性的關(guān)鍵基團,揭示了該類化合物與CK2α之間無序的相互作用誘導(dǎo)CK2α柔性區(qū)域構(gòu)象改變的結(jié)構(gòu)機制,可為高活性CK2-ATP競爭性抑制劑的設(shè)計優(yōu)化提供理論指導(dǎo)。7-羥基香豆素衍生物也是一類CK2-ATP競爭性抑制劑,且因具有較強抗腫瘤細胞增殖和抗氧化生物活性,成為重要的抗癌先導(dǎo)化合物。為提高此類化合物的抑制活性,基于不同類別ATP競爭性抑制劑與CK2的結(jié)合模式,針對7-羥基香豆素骨架結(jié)構(gòu),定向鉸鏈區(qū)極性殘基,設(shè)計優(yōu)化并合成了氟代-7-羥基香豆素衍生物。生物活性評價結(jié)果表明,8-氯-7-羥基-4-三氟甲基香豆素具有較高的CK2抑制活性(IC50=0.4μM)和良好的抗肺癌細胞A549的增殖能力(IC50=20.15μM)。其抑制活性明顯優(yōu)于類似物8-氯-7-羥基-4-甲基香豆素(CK2抑制活性為IC50=2.2μM,A549抑制活性為IC50=29.26μM)。研究提出了針對7羥基香豆素類化合物的優(yōu)化修飾方案,獲得了兼具抗腫瘤細胞增殖能力和CK2激酶抑制活性的新型7-羥基香豆素衍生物,為以往單一作用模式CK2抑制劑的優(yōu)化改造提供了一定的理論和實驗指導(dǎo)。相對于傳統(tǒng)ATP競爭性抑制劑而言,新型環(huán)肽抑制劑(Pc)可有效干擾α與β亞基的相互作用(IC50=3.0μM),從而間接抑制CK2對底物的磷酸化。環(huán)肽熱點殘基(Tyr188和Phe190)的突變會導(dǎo)致環(huán)肽抑制活性的明顯降低(IC50100μM)。為了探究熱點殘基突變導(dǎo)致環(huán)肽抑制活性降低的結(jié)構(gòu)機制,分別對野生型體系和兩個突變復(fù)合物體系(Y188A和F190A)進行100納秒長時分子動力學(xué)模擬,并結(jié)合主成分分析、動態(tài)相關(guān)性矩陣和自由能計算等方法對三個體系進行了深入的分析。結(jié)果表明,環(huán)肽與CK2α間形成了有序的疏水和極性相互作用網(wǎng)絡(luò),氫鍵主要維持環(huán)肽結(jié)構(gòu)穩(wěn)定以及環(huán)肽與CK2α的結(jié)合定位,而極性和疏水作用主要維持環(huán)肽殘基與CK2α殘基間的相互作用。兩個突變體系中,有序的相互作用網(wǎng)絡(luò)均遭到了不同程度的破壞,從而導(dǎo)致環(huán)肽抑制活性降低。在Y188A體系中,環(huán)肽與CK2α之間的關(guān)鍵氫鍵消失以及疏水作用減弱導(dǎo)致了環(huán)肽活性的降低。在F190A體系中,環(huán)肽結(jié)構(gòu)穩(wěn)定性的降低以及環(huán)肽與CK2α之間的重要疏水作用消失,使得環(huán)肽對CK2抑制活性顯著降低。研究揭示了環(huán)肽關(guān)鍵殘基突變導(dǎo)致CK2α與環(huán)肽親和力下降的結(jié)構(gòu)機制,證明了環(huán)肽與CK2α之間的有序疏水和極性相互作用網(wǎng)絡(luò)是保證環(huán)肽抑制活性的關(guān)鍵,并成功識別了負責(zé)環(huán)肽結(jié)合與定位的關(guān)鍵作用殘基,可為高活性干擾CK2α/CK2β相互作用抑制劑優(yōu)化設(shè)計提供一定的指導(dǎo)。綜上所述,論文采用多種分子模擬方法深入系統(tǒng)地探究了ATP競爭性抑制劑以及阻斷CK2α/CK2β相互作用抑制劑的結(jié)合模式和作用機理,提出了抑制劑的結(jié)構(gòu)優(yōu)化修飾方案,獲得了兼具抗腫瘤細胞增殖能力和CK2激酶抑制活性的新型7-羥基香豆素衍生物。上述研究為靶向CK2-ATP抑制劑先導(dǎo)化合物,以及CK2α/CK2β相互作用抑制劑的研發(fā)提供了理論支持和實驗指導(dǎo)。
[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.

【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R91

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