人痘苗病毒H相关磷酸酯酶VHR小分子抑制剂的发现

发布时间：2018-01-16 08:20

本文关键词：人痘苗病毒H相关磷酸酯酶VHR小分子抑制剂的发现　出处：《华东师范大学》2007年硕士论文　论文类型：学位论文

【摘要】： 人痘苗病毒H1相关磷酸酯酶(Human vaccinia H1-related phosphatase，VHR)是非典型的双位点特异性磷酸酯酶(dual-specificity phosphatase，DSPs)家族中的一员。VHR仅有催化结构域，是研究DSPs结构与功能的理想模型。它对有丝分裂原激活蛋白激酶(mitogen-activated protein kinases，MAPK)家族成员胞外信号调控激酶(extracellular signal-regulated kinases，ERK)和c-Jun氨基末端激酶(c-Jun N-terminal kinases，JNK)进行去磷酸化，是MAPK信号转导途径的负调节者。最近研究发现，VHR在众多的肿瘤细胞株中高表达，并且通过负调控MAPK信号通路，在肿瘤细胞的生长、分化及衰老方面起到重要作用，VHR因此成为潜在的药物作用靶分子。我们从原核大肠杆菌系统中成功表达并纯化得到VHR重组蛋白，通过对VHR的一系列酶学特性，包括DTT、EDTA和盐离子浓度的影响、pH依赖性、动力学分析及阳性抑制剂(Na_3VO_4)抑制作用分析，并优化VHR的活性检测方法，建立了VHR的高通量筛选(high throughput screening，HTS)模型。通过对国家化合物样品库中的65760个样品进行筛选，得到了34个抑制活性较好的化合物，其IC_(50)均低于3μg／mL。其中活性化合物Comp 1的IC_(50)为1．20μM，为结构新颖的VHR小分子抑制剂。通过分子水平酶动力学方法对其抑制性质进行研究，发现该化合物为可逆的、非竞争型VHR抑制剂。通过检测Comp 1对其它蛋白酪氨酸磷酸酯酶(Protein tyrosine phosphatases，PTPs)的选择性，发现Comp 1对经典跨膜受体型PTPs，如白细胞共同抗原(leukocyte common antigen，LCA or CD45)和白细胞共同抗原相关蛋白(leukocyte common antigen-related protein，LAR)，对经典胞内非受体型PTPs，包括蛋白酪氨酸磷酸酯酶1B(Protein Tyrosine Phosphatase 1B)和含有SH2结构域的蛋白酪氨酸磷酸酯酶1(SH-2 domain containing tyrosine phosphase 1，SHP1)，对同属于双位点特异性磷酸酯酶亚家族的细胞分裂周期25同源物A(cell division cycle 25 homolog A，CDC25A)均具有较高的选择性，表现出极低的抑制活性；而对含有SH2结构域的蛋白酪氨酸磷酸酶2(SH-2 domain containing tyrosine phosphase 2，SHP2)和细胞分裂周期25同源物B(cell division cycle 25 homolog B，CDC25B)具有一定的选择性，表现出较强的抑制活性。另外，我们对Comp 1细胞水平的生物活性进行了评价，发现Comp 1能提高宫颈癌细胞系Hela细胞中ERK和JNK的磷酸化水平，并能抑制宫颈癌细胞系HeLa、结肠癌细胞系HT-29、乳腺癌细胞系MDA-MB-435S的生长。高剂量的Comp 1可诱导HeLa细胞周期阻滞在G2／M期，并诱导出现明显的细胞凋亡。这一高活性、高选择性的新型小分子VHR抑制剂的发现、相关抑制性质的研究以及在细胞水平的生物活性评价，为其进一步结构优化和药理学、药效学研究打下坚实的基础，这对于进一步理解选择性所依存的分子机制及寻找高效特异的PTPs抑制剂有着重要的意义，，同时也为更加广泛地研究VHR的生理及病理功能提供了有力的工具。
[Abstract]:Human vaccinia H1-related phosphatase. VHRs are atypical dual-specificity phosphatase. VHR, a member of the DSPs family, has only a catalytic domain. It is an ideal model to study the structure and function of DSPs, which acts on mitogen-activated protein kinases. Extracellular signal-regulated kinases. ERK) and c-Jun N-terminal kinase (c-Jun N-terminal kinases-JNKK) were dephosphorylated. It is a negative regulator of MAPK signal transduction pathway. Recently, it has been found that VHR is highly expressed in many tumor cell lines and grows in tumor cells through negative regulation of MAPK signaling pathway. VHR plays an important role in differentiation and aging. The recombinant VHR protein was successfully expressed and purified from prokaryotic Escherichia coli system. The recombinant protein was expressed and purified by a series of enzymatic properties of VHR, including the concentration of VHR and salt ions. The pH dependence, kinetic analysis and inhibitory effect of VHR were analyzed, and the activity of VHR was optimized. The high throughput screening of VHR with high throughput screening was established. HTS) model. 34 compounds with good inhibitory activity were obtained by screening 65760 samples from the national compound sample bank. The ICs _ (50) of the active compound Comp _ 1 were less than 3 渭 g / mL and 1.20 渭 M. The inhibition properties of VHR were studied by enzyme kinetic method at molecular level. It was found that the compound was reversible. The protein tyrosine phosphatases of other protein tyrosine phosphatase (Comp 1) was detected by Comp 1 as a non-competitive VHR inhibitor. The selectivity of Comp 1 to classical transmembrane receptor PTPs, such as leukocyte common antigen, was found. LCA or CD45) and leukocyte common antigen-related protein. Larix, for classical intracellular non-recipient PTPs. These include protein tyrosine phosphatase 1B, protein Tyrosine Phosphatase 1B, and protein tyrosine phosphatase 1, which contains the SH2 domain. SH-2 domain containing tyrosine phosphase 1. SHP1A, a cell division cycle 25 homologue belonging to the double locus specific phosphatase subfamily, was identified as Agncell division cycle 25 homolog A. CDC25A) showed high selectivity and very low inhibitory activity. The protein tyrosine phosphatase 2H 2 domain containing tyrosine phosphase 2 contained SH2 domain. SHP2and BU cell division cycle 25 homolog CDC25B) have some selectivity. It showed strong inhibitory activity. In addition, we evaluated the biological activity of Comp 1 cells and found that Comp 1 could increase the phosphorylation of ERK and JNK in Hela cells. It can also inhibit the cervical cancer cell line HeLaand the colon cancer cell line HT-29. The growth of breast cancer cell line MDA-MB-435S. High dose of Comp 1 could induce HeLa cell cycle arrest in G 2 / M phase and induce obvious apoptosis. The discovery of this highly active and highly selective new small molecule VHR inhibitor, the study of its related inhibitory properties, and the evaluation of its biological activity at the cell level are further structural optimizations and pharmacology. The study of pharmacodynamics lays a solid foundation for further understanding the molecular mechanism of selectivity and searching for highly efficient and specific PTPs inhibitors. It also provides a powerful tool for studying the physiological and pathological functions of VHR more widely.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373

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