小分子阳离子抗菌肽虚拟组合设计、筛选与效应的研究

发布时间：2018-06-24 10:21

本文选题：抗菌肽 + LfcinB64-9虚拟组合设计　；参考：《重庆理工大学》2012年硕士论文

【摘要】：针对当前抗菌药物耐药性强的问题，特别是抗生素的滥用日趋严重，人类亟待开发出一类新型、高效、低毒、无残留的抗菌药物。而抗菌肽具有抗菌谱广、稳定性好、副作用小、无耐药性等优点，有望克服当前抗菌药物的耐药性强、副作用大等系列难题。目前，多种分子设计与改造方法用于抗菌肽研究，期望在提高抗菌活性同时进一步降低毒副作用，进而为抗菌肽的临床应用奠定基础；尽管现有抗菌肽的分子改造取得了可喜进展，但在抗菌活性、化学合成、细胞毒性等方面还存在诸多问题。基于上述研究现状，本文提出全新“肽链短、电荷高、两亲性强、优势氨基酸多”的“小分子“”阳离子”抗菌肽的设计理念和虚拟组合设计方法。以阳离子抗菌肽LfcinB64-9(RRWQWR)为模板，基于抗菌肽模式序列要求、优势位点的氨基酸结构及其化学修饰，开展抗菌肽的虚拟组合设计，并通过定量构效关系实现其高通量筛选，具体如下：①对现有抗菌肽数据库(APD2)中的抗菌肽进行一级序列比对分析，确定阳离子抗菌肽的序列模式；②对现有阳离子抗菌肽进行定量构效关系研究，确定核心序列的优势氨基酸及其化学修饰方法；③以LfcinB64-9为模板，基于序列模式、优势位点的氨基酸及其化学修饰，虚拟产生结构多样的抗菌肽库；④基于构效关系分析及拟肽二级结构筛选进行合成；⑤采用Fmoc固相方法合成拟肽、RP-HPLC纯化并质谱鉴定；⑥拟肽与LfcinB64-9通过抗菌活性、细胞毒性、稳定性考查筛选所设计的最好抗菌肽，为下一步的成药奠定基础。本文主要的研究成果是：(1)建立小分子阳离子抗菌肽的组合虚拟筛选路线：(2)成功的筛选到三条高效、低毒、副作用小、无耐药性的小分子阳离子抗菌活性多肽。实验结果表明“肽链短、电荷高、两亲性强、优势氨基酸多”的小分子阳离子抗菌肽的设计理念和虚拟组合设计方法，节约了新型抗菌肽设计与筛选的时间和成本，并提高了抗菌肽分子设计的合理性和可行性。本文所设计、筛选的三条抗菌肽有望成为结构特异性好、体内抗酶解能力强、作用机理明确、毒副作用小、化学合成容易、具有成药潜力的小分子阳离子抗菌肽。总而言之，基于组合肽库与分子模拟方法开展抗菌肽的虚拟组合设计与高通量筛选研究，不仅可以基于构效关系研究成果合理设计结构多样性的多肽分子，，并有效提高筛选效果、降低研究成本；有望进一步提高其抗菌活性并解决规模生产问题，对抗菌肽走向实际临床应用具有重要理论研究价值。
[Abstract]:In view of the problem of strong antimicrobial resistance, especially the serious abuse of antibiotics, it is urgent to develop a new class of antimicrobial drugs with high efficiency, low toxicity and no residue. However, antimicrobial peptides have the advantages of wide antibacterial spectrum, good stability, small side effects and no drug resistance, which are expected to overcome the current antimicrobial drug resistance, side effects and other problems. At present, many molecular design and modification methods are used in the study of antimicrobial peptides, in order to improve the antibacterial activity and further reduce the toxic side effects, thus laying a foundation for the clinical application of antimicrobial peptides. Although the molecular modification of antimicrobial peptides has made great progress, there are still many problems in antibacterial activity, chemical synthesis, cytotoxicity and so on. Based on the above research status, this paper presents a new "small molecule" cationic "antimicrobial peptide design method with" short peptide chain, high charge, strong amphiphilicity and many dominant amino acids ". The cationic antimicrobial peptide LfcinB64-9 (RRWQWR) was used as the template. Based on the requirements of the antibacterial peptide pattern sequence, the amino acid structure of the dominant site and its chemical modification, the virtual combination design of the antimicrobial peptide was carried out, and its high throughput screening was realized by quantitative structure-activity relationship. The following is the first order sequence alignment analysis of antimicrobial peptides in the existing antimicrobial peptide database (APD2), and the quantitative structure-activity relationship of existing cationic antimicrobial peptides is determined by determining the sequence pattern of cationic antimicrobial peptides. To determine the dominant amino acids of core sequences and their chemical modification methods: using LfcinB64-9 as template, based on sequence patterns, amino acids at dominant sites and their chemical modification, the antibacterial peptide libraries with various structures were produced. 4 based on the structure-activity relationship analysis and the screening of the secondary structure of the mimic peptide, the synthesis method was used to purify the peptide by FMOC solid phase method by RP-HPLC and to identify the antibacterial activity and cytotoxicity of LfcinB64-9 and the peptide by mass spectrometry. Stability test and screening of the best antimicrobial peptides designed to lay the foundation for the next step of proprietary medicine. The main results of this paper are as follows: (1) to establish a virtual screening route for small cationic antimicrobial peptides; (2) to successfully screen three small cationic antimicrobial peptides with high efficiency, low toxicity, small side effects and no drug resistance. The experimental results show that the design concept and virtual combination design method of small cationic antimicrobial peptides with short peptide chains, high charge, strong amphiphilic properties and more dominant amino acids can save the time and cost of the design and screening of new antimicrobial peptides. The rationality and feasibility of antimicrobial peptide design were improved. The three antimicrobial peptides designed in this paper are expected to be small cationic antimicrobial peptides with good structural specificity, strong anti-enzymatic ability in vivo, clear mechanism, small toxic side effects and easy chemical synthesis. In a word, virtual combinatorial design and high-throughput screening of antimicrobial peptides based on combinatorial peptide library and molecular simulation methods can not only reasonably design polypeptide molecules with diverse structures based on the results of structure-activity relationship research. It is expected to further improve its antibacterial activity and solve the problem of large-scale production, which has important theoretical research value for the practical clinical application of antimicrobial peptides.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【相似文献】