基于非天然氨基酸的高选择性小分子抗菌拟肽合理设计与构毒关系研究

Antimicrobial peptide (AMP) has numerous advantages, such as broad antimicrobial spectrum and low drug resistance, but the AMP-specific toxicity hinders its practical uses severely. Therefore, promoting the selectivity of AMP is the basis for its clinic applications. In this study, the small peptoids is explored for their innate toxicity. First, the scheme of rational design and high throughout screening of antimicrobial peptoids will be explored, which is based on the quantitative structure-activity relationship modeling of natural AMPs and their analogues, and the theoretical model of membrane-based selectivity for the peptoid will be constructed, which is based on the molecular modeling of the interaction between the peptoid and the membrane of bacterial and eukaryotic cell. Secondly, the active peptoids are screened by the experiments of stability and antimicrobial activity, and the mechanism of membrane-targeting is validated through the membrane lipid flip-flop and penetration methods. Finally, the experiments of dye leakage of lipid membrane, hemolysis and cytotoxicity are used to evaluate the selectivity and safety of the active peptoids. This study may establish the technical scheme of the rational design, theoretical prediction and high performance screening for the AMP and its analogues，and the active peptoids with high antimicrobial activity, stability, selectivity, and low drug resistance would be screened. Meanwhile, the theoretical models of structure-activity and structure-toxicity of the peptoid will be built, which are used to explore the mechanism of antimicrobial activity and general rule of AMP-specific toxicity. In general, this study will becomes the basis of research and development of antimicrobial peptoids with selectivity

抗菌肽(AMP)具有抗菌谱广、耐药性低等系列优点，但开发利用受其特异性毒性制约，因而提高选择性是解决其临床应用的关键。本研究针对AMP的特异性毒性，以小分子抗菌拟肽为研究对象，基于天然AMP及其类似物的构效关系研究，建立理论预测模型指导抗菌拟肽的合理设计与虚拟筛选；应用分子模拟方法分析拟肽与真核及细菌细胞膜间的相互作用差异，构建拟肽对细胞膜选择性作用的理论预测模型；通过稳定性与抗菌实验筛选活性拟肽分子，基于膜脂质翻转与去极化实验，验证拟肽以细胞膜为靶标的作用机制；采用脂质膜染料渗漏、体外溶血及细胞毒性实验，评价拟肽的选择性。本研究不仅可为拟肽的合理设计与高效筛选建立完整技术方案，筛选获得抗菌活性强、稳定性好、选择性高、耐药性低的活性拟肽分子，而且能够基于拟肽的构效与构毒关系研究结果，从分水平揭示拟肽的抗菌作用机制及其产生特异性毒性的本质与一般规律，为高选择性抗菌拟肽的研究与开发奠定基础。

鲍曼不动杆菌(AB)是一种严重的医源性病原菌，也是耐药发生率最高的革兰氏阴性菌。截至目前，多数耐药AB（MRAB）对美罗培南等药物耐药，是临床耐药菌感染治疗的难题。抗菌肽（AMP）具有抗菌谱广、杀菌快速、耐药性低等优点，有望开发成为一类新型抗菌药物。本研究开展AMP定量构效关系研究、活性测定、安全性与稳定性评价及作用机制研究，取得如下研究结果： .1. 使用SWR-MLR与OMP-MLR方法建立了金黄色葡萄球菌和大肠杆菌的理论预测模型，其模型具有良好的可靠性(S. aureus: R2=0.93 vs R2=0.90, E. coli: R2=0.84 vs R2=0.70)和预测能力(S. aureus: Q2=0.85 vs Q2=0.75, E. coli: Q2=0.72 vs Q2=0.70)；保守残基分析与优势氨基酸贡献对AMP设计具有指导作用。.2. 合成AMP结构正确，纯度达到95%以上；天然小分子AMP合成容易、成本低；而结构修饰物合成难度大、成本高。.3. 抗菌肽对耐药菌的MIC在8-64 μg/mL之间，特别是对MRAB具有良好抑菌作用（MIC为8-32 μg/mL）；AMP的起效速度与持续时间均优于亚胺培南，且与亚胺培南有协同作用。.4. AMP在血浆中稳定性良好，90分钟后仍有约50% AMP保留；但高浓度CaCl2和NaCl溶液会降低其抗菌效果。.5. 分子AMP（512μg/mL）的体外溶血率低于10%，但碳-端苯乙胺修饰产物可能增强抗菌肽溶血毒性；部分AMP具有抑制肿瘤细胞生长作用，浓度256μg/mL时对HepG2的抑制率高达80%以上。.6. 仪器观察发现AMP可破坏细菌细胞膜，改变通透性；小分子AMP主要作用于细胞膜，对脂质膜破坏能力强，而长链AMP则能穿过细胞膜达到胞内；对细胞膜的破坏能力较弱。 .7. AMP可与DNA能够有效结合，而且与抗菌活性相关，表明AMP除作用于细胞膜外，还能作用于胞内DNA。.综上所述，本项目完成了小分子AMP的理论预测模型构建、设计与虚拟筛选，活性测定与稳定性考察，溶血毒性与肿瘤生长抑制评价，证实AMP以细胞膜和DNA为靶标的作用机制等相关研究，筛选获得6个对MRAB具有良好抑菌活性的小分子AMP，为AMP的应用开发奠定了物质基础。

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