【作者】 黄惠明；

【导师】 刘成梅；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2010， 博士

【摘要】 氨肽酶N(APN,又称CDl3)是含锌离子的一种膜结合型外肽酶,为依赖锌离子的金属蛋白酶系列,在哺乳动物的小肠肾脏、中枢神经系统等组织中有广泛表达。APN大量表达于肿瘤细胞表面,对肿瘤的血管生成、侵害具有重大的作用。该酶降解细胞外基质的主要成分,从而促进肿瘤细胞在人体的转移和生长；另还能够对许多免疫活性物质进行降解,削弱NK细胞和巨噬细胞识别和杀伤肿瘤细胞的能力,降低机体的免疫力。故抑制APN的活性可以控制对肿瘤肿瘤细胞血管生成,同时增强粒细胞的趋化性,使机体的免疫力得以提高。APN已被证明作为人体冠状病毒TEGV和229E病毒的受体。所有这些发现使APN的研究成为一个关注的目标,同时,近年来抗肿瘤药物研发的热点趋向于开发高选择性、高效的的APN抑制剂。基于结构基础的药物设计原理和对已知的类肽类APN抑制剂结构特征的分析与总结,化合物与APN的结合部位可分为三个部分,A部分是同Bestatin的苯环相互作用的疏水口袋,B部分是锌结合组(ZBG型),C部分是另一侧疏水口袋。通过对计算机辅助药物设计软件的应用设计新的药物,结合酶的晶体结构,本研究设计并合成了酒石酸、苹果酸和烟酸三个系列共40个结构全新的化合物。其化学结构由波谱分析(IR、ESI-MS、1H NMR)确认,所合成的目标化合物经查阅文献证实,未见文献报道,均为新化合物。对所合成化合物进行了体外抑酶实验,并对其活性作出初步评价。选择APN来对合成物进行筛选,从而确定其酶活性的大小。其结果表明：本实验设计合成的化合物中的大多数都对APN有抑制作用。实验结果表明,L-酒石酸系列化合物对APN抑制活性最高,其半数抑制浓度(IC50)达到微摩尔级,具有较高抑酶作用。本实验选用人红细胞白血病K562细胞对所合成的化合物进行抗肿瘤活性筛选,利用MTT法间接测定各目标产物作用后肿瘤细胞增殖的变化,并计算出肿瘤细胞的抑制率。并且,从所合成化合物选出四个肿瘤细胞抑制率较好的化合物(A1、B1、C1、D2),计算出它们的半数抑制浓度(IC50),筛选出体外活性较好的化合物D2。更多还原

【Abstract】 Aminopeptidase N (APN/identical to CD 13) belongs to a family of zinc depen-dent exopeptidase has become an attractive target for structure-based drug designing in the last decades. It was originally used as a marker for the myeloid differentiation antigen.Aminopeptidase N (APN) is expressed on various mammalian cells including monocytes, myeloid, epithelial cells of the intestine and kidney, fibroblasts, endoth-elial cells and tumor cells. APN has been also proved to behave as a receptor for corona-viruses TEGV and 229E in humans.More impotantly, many researches have shown that APN is involved in the regulation of tumour cell invasion and metastasis. In addition, APN is involved in the processes of immunological regulation, and as a result, many immunologic active materials have been degradated and the ability of macrophage and NK cells to identify and kill tumor cells have been weakened. All these findings make this enzyme an interesting target for possible anti-tumor drugs research, which require the development of potent and more selective inhibitors.Based on the infrastructure of design principles, analysis and summary the structural features of peptide-like APN inhibitors which have been synthesized. Studies have discovered that the binding site of the APN with compouns can be divided to three parts, part A is a hydrophobic pocket which interacts with the phenyl group of Bestatin; part B is the zinc binding group (ZBG) and part C is a hydrophobic pocket on the other side. In this thesis, by the combination of crystal structure of the enzyme and application of computer-aided drug design software, we designed and synthesized three series of 40 novel peptide-like derivatives:tartaric acid derivatives, malic acid derivatives and nicotine acid derivatives. Their structures are confirmed by IR, ESI-MS and 1H NMR. All the targeted compounds are novel without any report by now.Preliminary bioactivity assays are carried out in vitro. The target compounds are evaluated for inhibitory activities toward APN. As a result, most of these newly syn-thesized compounds show good inhibitory activities on APN. The results of experi- ment show that L-tartaric acid derivatives are the most activity inhibitors toward APN, their half inhibitory concentration (IC50) achieved to micromolar range, these new compounds have potent and selective inhibitory activities toward APN.The newly synthesized compounds were evaluated for their in vitro cytotoxicity by growth-inhibition studies using erythrocyte leukeemic cell line (K562). MTT assay was used of them and calculate the rate of inhibition against the tumor cells. Of these compounds, compounds A1,B1,C1 and D2 are the most active compounds. So we choose them as experimental subject, continued to obtain their cytotoxic activity IC50 and we found compound D2 have the best inhibitory activity toward K562 cells.更多还原