蛋白质酪氨酸磷酸酶家族不可逆苯并咪唑噻唑衍生物抑制剂的发现

发布时间：2018-09-11 16:54

【摘要】：研究背景蛋白质酪氨酸磷酸化修饰是真核细胞内信号转导的一种重要调控,动态可逆的蛋白质酪氨酸磷酸化程度由蛋白质酪氨酸激酶(PTK)和蛋白质酪氨酸磷酸酶(PTP)协同调控。PTP在许多生理过程中发挥关键作用,调控细胞的增殖分化、迁移、细胞内代谢、免疫应答和神经活动等。异常的PTP活力可能引发多种人类疾病,如癌症或者糖尿病。PTP1B在胰岛素信号通路中起负调控作用,其活性异常与糖尿病发生有关。YopH是耶尔森菌逃避宿主先天免疫应答的重要因子;纹状体富集蛋白质酪氨酸磷酸酶(Striatal enriched tyrosine phosphatases,STEP)作为一种中枢神经系统特有的磷酸酶,对突触的可塑性、神经元的生存等具有重要调节作用。STEP功能失调有可能导致包括阿尔兹海默症在内的多种神经系统退行性性疾病。目前,PTP作为潜在的药物靶标日益引起关注。特异性调控PTP的小分子化合物不仅是应用于开发临床药物的希望所在,也可作为特定的活性探针来研究并阐明PTP在细胞内信号转导中发挥的重要作用。因此发展高效的小分子探针并深入研究其作用机制具有重要意义。研究目的通过获得纯化的STEP、LYP、PTP1B、YopH和MEG2蛋白质酪氨酸磷酸酶进行酶学分析及小分子抑制剂活性筛选的研究。材料与方法将STEP、LYP、PTP1B、YopH和MEG2蛋白磷酸酶野生型序列亚克隆到pET-15b、pET-28a和pET-22b等表达载体上,随后转化至E.coli BL21工程菌中表达,通过亲和层析和凝胶阻滞层析等技术,获得纯化的STEP、LYP等磷酸酶,并以pNPP为底物对其进行体外酶活测定,并运用连续读数法、IC50半抑制效率测定法、抑制剂酶动力学测定进行抑制剂筛选,随后通过测定抑制剂对其他磷酸酶PPM家族的选择性揭示化合物是否具.有特异性,确定化合物的作用。结果实验结果表明STEP、YopH、MEG2等磷酸酶蛋白表达成功,并可经纯化活动纯度超过90%的酶用于体外酶学实验。纯化后的STEP、YopH、MEG2等磷酸酶SDS-PAGE电泳表观分子量分别为35kDa、33kDa、38kDa等。随后,根据对STEP等磷酸酶基本酶活分析,又从化合物库中筛选出1种具明显抑制作用且结构新颖的抑制剂,小分子化合物E4以时间和浓度依赖性的方式抑制了 STEP蛋白活性。进一步研究表明,化合物E4以时间依赖性方式抑制一系列PTP,而其较少抑制或没有抑制金属依赖性蛋白磷酸酶。结论在这里,我们将化合物E4表征为一类新的PTP活性探针。通过活性依赖方式共价标记在PTP的活性位点的化学探针可以极大的促进对PTP功能研究。总的来说,这种新鉴定的PTP的共价抑制剂可以被用来作为PTP活性位点Cys的定向探针来研究PTP在细胞信号传导中的作用。
[Abstract]:Background protein tyrosine phosphorylation modification is an important regulation of signal transduction in eukaryotic cells. The dynamic and reversible protein tyrosine phosphorylation is coordinated by protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP), which plays a key role in many physiological processes and regulates cell proliferation, differentiation, migration and intracellular metabolism. Immune response and neural activity, etc. Abnormal PTP activity may lead to a variety of human diseases, such as cancer or diabetes. PTP1B plays a negative role in insulin signaling pathway. The abnormal activity of PTP is related to the occurrence of diabetes. YopH is an important factor that Yersinia evades the host innate immune response. Striatum enriched protein tyrosine phosphatase (Striatal enriched tyrosine phosphatases,STEP) as a central nervous system specific phosphatase, synaptic plasticity, Neuron survival and other important regulatory effects. Step dysfunction may lead to a variety of neurodegenerative diseases, including Alzheimer's disease. At present, PTP as a potential drug target has attracted more and more attention. Small molecular compounds that specifically regulate PTP are not only promising for the development of clinical drugs, but also serve as specific active probes to study and elucidate the important role of PTP in intracellular signal transduction. Therefore, it is of great significance to develop high efficient small molecular probes and to study the mechanism of their action. Objective to study the enzymatic analysis of purified STEP,LYP,PTP1B,YopH and MEG2 protein tyrosine phosphatase and the screening of small molecule inhibitor activity. Materials and methods STEP,LYP,PTP1B,YopH and MEG2 protein phosphatase wild-type sequences were subcloned into pET-15b,pET-28a and pET-22b expression vectors, and then transformed into E.coli BL21 engineering bacteria for expression. Purified STEP,LYP and other phosphatase were obtained by affinity chromatography and gel block chromatography. The enzyme activity in vitro was determined by using pNPP as substrate, and the IC50 semi-inhibition efficiency was determined by continuous reading method, and the inhibitor was screened by enzyme kinetic assay. The selectivity of inhibitors to other phosphatase PPM families was then determined. Have specificity, determine the action of compound. Results the results showed that STEP,YopH,MEG2 and other phosphatase proteins were successfully expressed and could be used in enzymatic experiments in vitro after purified enzyme with activity purity of more than 90%. The apparent molecular weight of purified STEP,YopH,MEG2 isophosphatase SDS-PAGE was 35kDaA33kDa / 38kDa, respectively. Then, according to the analysis of the activity of phosphatase such as STEP, a novel inhibitor with obvious inhibitory effect was selected from the compound library. The activity of STEP protein was inhibited by small molecular compound E4 in a time-and concentration-dependent manner. Further studies have shown that compound E4 inhibits a series of PTP, in a time-dependent manner, but it has little or no inhibition of metalloprotein phosphatase. Conclusion compounds E4 are characterized as a new PTP active probe. The study of the function of PTP can be greatly promoted by covalently labeling the chemical probes at the active sites of PTP in an activity-dependent manner. In general, this newly identified covalent inhibitor of PTP can be used as a directional probe for Cys, the active site of PTP, to study the role of PTP in cell signal transduction.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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