【作者】 史喆；

【导师】 施蕴渝； 吴季辉；

【作者基本信息】 中国科学技术大学 ， 生物化学和分子生物学， 2007， 博士

【摘要】 本论文的工作重点是结合计算机虚拟筛选和核磁共振（NMR）技术筛选人双特异性蛋白质磷酸酶VHR的抑制剂，发现了一个结构新颖的强效抑制剂，并进一步研究了此抑制剂在细胞内的影响。VHR在调控MAPK级联途径中起重要作用，从而与许多人类疾病联系在一起，因而抑制剂的发现不仅有助于蛋白质酪氨酸磷酸酶生理功能的阐明，而且有作为药物先导物的前景。论文共分为以下四个部分：第一章简介了计算机辅助药物设计和筛选，其中重点综述了利用分子对接方法虚拟筛选小分子化合物库的原理，以及一些常用分子对接程序的理论基础和筛选流程。虚拟筛选是创新药物研究的新方法和新技术，并且已经成为一种与高通量筛选互补的实用化工具，加入到了创新药物研究的工作流程中。随着结构生物学、结构基因组、功能基因组学及蛋白质组学的发展，越来越多的生物大分子三维结构被测定，虚拟筛选能够发挥更大的作用。分子对接是药物发现和设计中一种非常重要的方法，将会减少随机筛选的盲目性，大大加快药物发现的速度。第二章主要综述了核磁共振（NMR）技术在药物筛选中的方法与应用。核磁共振（NMR）技术在方法学和硬件上的迅速发展，使其成为化学和结构生物学中最重要的波谱技术。特别是生物核磁共振技术在药物研发过程中，提供从药物设计结构信息到基于NMR技术进行配体的筛选的广泛应用，使得生物核磁共振技术在药物研究领域地位不断加强，成为药物研究的基础工具。核磁共振已经作为一个强大的筛选方法来识别和探测蛋白和配体的相互作用。药物公司也已经将NMR筛选平台纳入药物发现和药物设计部门。NMR筛选可以被定义为通过观察当蛋白和配体相互作用时NMR参数的变化来识别靶蛋白的小分子配体。NMR筛选可以被分为基于探测大分子蛋白的参数变化和基于探测小分子配体的参数变化两大类。对于大分子，可观测的参数被限制只能是最典型的化学位移。对于小分子，可选择的参数就比较多。包括纵向，横向驰豫，扩散系数，分子间和分子内磁化转移。后者包括转移NOE（Nuclear Overhauser Effect），NOE pumping和反向NOE pumping，WaterLOGSY实验。第三章介绍了结合计算机虚拟筛选和核磁共振（NMR）技术筛选人双特异性蛋白质磷酸酶VHR的抑制剂的研究。首先对VHR的功能和结构，已发现的抑制剂进行了全面介绍。并进一步研究了抑制剂在细胞内的影响。双特异性蛋白质磷酸酶是参与促有丝分裂信号转导和细胞周期调控的主要酶类。既能从含有磷酸酪氨酸，又能从含有磷酸苏氨酸／丝氨酸的蛋白质中移去磷酸盐。VHR在调控MAPK级联途径中起重要作用，从而与许多人类疾病联系在一起。ZAP-70酪氨酸激酶和牛痘相关激酶VRK 3能通过不同方式增强VHR的活性，从而促进VHR对Erk2-Elk-1途径的抑制。VHR能够调节细胞周期而且能够调节自身的表达量．缺失VHR的细胞会在G1-S和G2-M期静息，并且有衰老迹象。我们首先运用分子对接程序DOCK4.0对小分子库进行虚拟筛选，通过扩散编辑NMR方法评估所选小分子的结合能力，发现了4个能够结合VHR的配体。并以对硝基苯酚磷酸盐为底物测定计算了它们抑制VHR去磷酸的能力，发现其中一个小分子GATPT具有新颖的结构，强烈并且竞争性抑制VHR的酶活(IC₅₀=2.92μM)。AutoDock计算显示的复合物结构模型表明这个小分子和蛋白质活性口袋紧密结合。一系列实验结果显示抑制剂GATPT能够影响细胞内VHR的天然底物phosphor-ERK和phosphor-JNK的表达量。而且它对细胞周期的影响和RNA干扰细胞内VHR表达所导致的细胞周期变化一致。VHR的小分子抑制剂可以为研究蛋白质酪氨酸磷酸酶生理功能提供一种新的途径。第四章首先对本工作的研究背景，即AF-6蛋白及PDZ结构域的功能和结构、Bcr蛋白的功能和结构进行了简单的介绍。AF-6基因最早是在急性淋巴性白血病人中作为ALL-1的融合配体被发现的。AF-6在细胞连接和信号转导中发挥双重作用。AF-6 PDZ结构域位于AF-6蛋白的C-端部分。在细胞中AF-6蛋白参与的很多蛋白质—蛋白质相互作用是由它的PDZ结构域介导的。细胞连接和肿瘤的浸润转移关系密切，上皮细胞向间质细胞的转化过程中细胞结构改变，失去极性，并且失去与邻近细胞的接触，获得运动性。因此抑制细胞黏附的小分子有可能发展成阻止肿瘤转移的药物。基于本组解出AF-6 PDZ结构域NMR溶液结构和AF-6蛋白质PDZ结构域与Bcr C-末端小肽的复合物NMR溶液结构，我们通过计算虚拟筛选化合物库和荧光标记蛋白来找出能够和AF-6 PDZ结构域的两个小分子，并希望以此为基础进一步优化增强小分子配体的结合能力，从而为研究AF-6蛋白质PDZ结构域在细胞内的功能提供一种途径。更多还原

【Abstract】 Our work focuses on identifying the inhibitor of Human Vaccina H1 related protein (VHR), by using virtual screening and NMR method. One compound with novel structure was found to be a competitive and potent inhibitor of VHR. And the effects of this inhibitor in cells were also investigated. VHR is a dual-specific phosphatase (DSPs) playing an important role in the mitogen-activated protein (MAP) kinase cascades regulation. It is also a potential drug target for designing inhibitors against diseases related to the immune response. The inhibitor may be contribute to the clarification of physiological function of VHR, and may be a promising candidate for designing of more effective agents against VHR.First chapter introduces and reviews the current docking methods in drug virtual screening. As a new method or technology for drug discovery, virtual screening has been involved into the pipeline of drug discovery and development as a practical tool. With the development of structural biology, structural genomics, functional genomics, and proteomics, more and more three dimensional structures of biologic molecule will be expressed, virtual screening would play a vital role which could accelerate the course of drug discovery.The second chapter is the brief review of NMR screening techniques in drug discovery and drug design. Library about NMR screening design is also discussed. Pharmaceutical and academic nuclear magnetic resonance (NMR) groups have implemented NMR screening techniques as a powerful approach to identify and to investigate protein/ligand interactions. Pharmaceutical groups in particular have incorporated NMR screening strategies into their drug discovery and drug design programs. This stems from the fact that NMR screening is naturally synergistic with combinatorial or medicinal chemistry, high throughput screening (HTS), structure-based drug design, and genomics.NMR screening can be defined as the identification of small molecule ligands for macromolecular targets by observation of a change in an NMR parameter that occurs upon their interaction. NMR screening methods can be divided into those that detect interactions by observation of either macromolecule NMR parameters or small molecule NMR parameters. In the case of macromolecules, the parameter that can be monitored is limited typically to chemical shifts. For small molecules, the choice of NMR parameters is more diverse. These include longitudinal, transverse; diffusion coefficients; and intermolecular and intramolecular magnetization transfer. The latter includes transferred NOE, NOE pumping and reverse NOE pumping, saturation transfer, and WaterLOGSY experiments.In the third chapter, we described the work about identifying the potent inhibitor of VHR. Human Vaccinia H1-related phosphatase (VHR) is a dual-specific phosphatase (DSPs) playing an important role in the mitogen-activated protein (MAP) kinase cascades regulation. It is also a potential drug target for designing inhibitors against diseases related to the immune response. Combining virtual and NMR-based ligand screening strategy, we successfully identified four VHR inhibitors, of which GATPT (glucosamine-aminoethoxy-triphenyl-tin) can bind to VHR with a K_i value of 2.54μM. The putative binding mode of GATPT was constructed by molecular docking simulation to provide structural insights into the ligand binding mechanism. Furthermore, we found that this compound can significantly inhibit the dephosphorylation of the extracellular regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) and block transition of G1-S phase in the cell cycle. Therefore, GATPT structure is a promising candidate for designing of more effective agents against VHR.The fourth chapter introduced the work processed on identifying inhibitors of AF-6 PDZ domain. The human AF-6 gene was initially discovered as a fusion partner of the ALL-1 gene in acute lymphoblastic leukemia. The human AF-6, a scaffold protein between cell membrane-associated proteins and actin cytoskeleton, plays an important role in special cell-cell junctions and signal transduction. Molecules that can block cell adhesion may have value as therapeutic agents that prevent tumor metastasis. Therefore, the inhibitors of AF-6 PDZ domain may be a potential candidate for blocking cell adhesion and preventing tumor metastasis.更多还原