【作者】 徐杰；

【导师】 陈婉蓉； 南发俊； 李佳； 李静雅；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 负责剪切新生多肽或蛋白N末端甲硫氨酸的甲硫氨酰氨肽酶(methionineaminopeptidase,MetAP)是一类二价金属离子依赖的蛋白酶,对维持细胞正常生理功能具有重要作用。甲硫氨酰氨肽酶是潜在的抗细菌、抗真菌和肿瘤治疗分子靶点。光亲和标记技术是分子生物学,细胞生物学,有机化学等学科有机结合发展的新技术。光亲和标记利用光活性小分子化合物作为工具探针,经特定波长的光照射分解产生高活性中间体,与其受体活性部位形成不可逆的共价结合,其结果是在一个配体和它的特异性受体之间引入一个共价键,从而直接标记靶蛋白。光亲和标记技术在药物的发现与发展中主要有两个方面的应用:靶标蛋白与小分子配体作用模式的确定(微观)及新靶点的确定(宏观)。运用光亲和标记技术,大量的与疾病相关的靶点被发现、多种小分子与生物大分子作用的模式被确定,这些信息对合理药物设计具有非常重要的意义。本论文在筛选得到酵母I型甲硫氨酰氨肽酶抑制剂的基础上,考察这些活性化合物对酵母菌的生长抑制。并采用光亲和标记技术对小分子化合物和大肠杆菌甲硫氨酰氨肽酶(Escherichia coli methionine aminopeptidase,EcMetAP 1) 的结合方式和作用机制做了初步的探讨。本论文第一部分的工作是对酵母I型甲硫氨酰氨肽酶(ScMetAP1) 的抑制剂进行生物活性测定,即抑菌实验。通过抑菌实验表明这类化合物对MetAp2缺失(△map2) 酵母比野生型(WT)酵母生长的抑制活性好,但是对MetAP1缺失(△map1) 酵母生长没有影响,说明这类化合物确实是选择性作用在ScMetAP1上,可作为潜在的抗真菌先导化合物。这类化合物对酵母生长的抑制活性与其在分子水平对Co(II)-ScMetAP1的抑制作用无明显相关性,这可能是因为体内ScMetAP1的金属离子不是CO2+,而是其他的二价金属离子。为了进一步探索这个问题,研究不同离子激活的ScMetAP1对化合物抑制选择性的变化,为ScMetAP1抑制剂的进一步改造和开发提供思路。这些实验结果表明金属离子替代的ScMetAP1改变了对抑制剂的选择性,同时表明,确定ScMetAP1内源性的金属离子对开发有疗效的ScMetAP1抑制剂具有重要意义。更多还原

【Abstract】 Methionine aminopeptidase (MetAP) carries out the important function of removing the initiator methionine residue from nascent peptides and proteins. MetAPs are potential targets for developing antibacterial, antifungal and anticancer candidates.Photoaffinity labeling is a newly developed technique, which is the combination of molecular biology, cell biology and organic chemistry. The method of photoaffinity labeling enables the direct labeling of target protein through a covalent bond which is photochemically introduced between a ligand and its specific receptor. Thus, photoaffinity labeling could be applied in two levels. If the binding site of a target protein with the particular pharmacophore is not defined, the photoaffinity labeling will give the structural information of receptor binding domain at micro-level. At macro-level, the method is useful for discovery of target protein from the evaluation of affinity by cross-linking. By photoaffinity labeling many proteins involved in diseases were found, and the ways interaction with lead compound were determined. These informations are very important for rational drug design.The work was. to detect the antifungal activity of some inhibitors of Saccharomyces cerevisiae methionine aminnopeptidase 1 (ScMetAP1) in vivo which were got by screening in vitro. And we investigated the interaction of PCAT series compounds with EcMetAP1 using photoaffinity labeling.The first part of this work was to detect the antifungal activity of inhibitors of ScMetAP1. These compounds prevented the growth of WT and Amap2 yeast, but has no influence on △mapl yeast. It suggested those compounds act on the target ScMetAPl in vivo.But no perfect relationship between the antifungal activity in vivo and the inhibitory effect on Co(II)-ScMetAP1 in vitro. It is possibly due to the metal ion of MetAP1 in yeast is not Co(II), but other divalent metal ions as Mn(II)、 Zn(II) or Fe(II). Next we investigated the inhibition on the different divalent metal activated ScMetAP1. The results indicated that these compounds have selective activity for ScMetAP1 activited by different divalent metal. It pave the way for finding anddesigning the potent inhibitior of ScMetAP1 in vivo.The second part of this work was to investigate the interaction of PCAT series compounds with EcMetAPl using photoaffinity labeling at micro-level. According to the structure -activity relationship of the compounds of PCAT, we designed and synthesized inhibitor of EcMetAP1 with Diazirine photoreactive group. This inhibitor was used as a probe and to label EcMetAPl. It was detected by Mass Spectrum that EcMetAP1 covalently labelled the probe successfully. After EcMetAP1/probe complex was digested by trypsin, the peptide covalent labelled with probe was separated and collected by HPLC. Molecular weight of the peptide is 1142 Da by mass spectrum (MS) detection and its sequence is deduced as "DGWTVK". The conclusion is PCAT pyridinium 3-substitute group binds into S1 pocket of EcMetAP1.更多还原