新型抗MRSA天然产物阿扎霉素F5a的绝对构型及细胞膜磷脂作用的分子机制研究

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to a variety of structural types of antibiotics, and the research and development of new anti-MRSA drugs is particularly urgent. Azalomycin F5a is a typical 36-membered macrolide produced by some streptomyces strains. After the relative configuration of azalomycin F5a was first assigned in 2012, it's remarkable anti-MRSA activity through acting on the cell membrane phospholipids of MRSA was discovered by us. While the molecular mechanism of azalomycin F5a against MRSA was not clear, and it's absolute configurarion was not assigned. Based on this, the absolute configuration of azalomycin F5a will be assigned by NMR method in this project. First, the specific derivatives and Mosher esters of degradation products derived from azalomycin F5a, together with the model compounds of special fragments, will be synthesized. Secondly, the absolute configurations of degradation products will be determined by NMR method and by comparing their NMR data with those of model compounds. Finally, the absolute configuration of azalomycin F5a will be assigned from those of degradation products. Simultaneously, combined with the biomembrane models respectively prepared by phosphatidylglycerol and the cell membrane lpids extracted from exponential phase MRSA, the molecular mechanism of azalomycin F5a acting on the cell membrane phospholipids will be researched by modern spectroscopic and biophysical techniques, such as raman spectroscopy, atomic force microscopy, differential scanning calorimetry and nuclear magnetic resonance, and by molecular dynamics simulation. Through above researches, a solid foundation will be established for the research and development of new anti-MRSA drug.

耐甲氧西林金黄色葡萄球菌（MRSA）对多种结构类型的抗生素相继耐药，新型抗MRSA药物的研发尤为迫切。天然产物阿扎霉素F5a为链霉菌代谢所产36元大环内酯的代表性化合物，申请人2012年首次报道其相对构型后，发现其具有显著的抗MRSA活性，且前期研究分析显示阿扎霉素F5a作用于MRSA的细胞膜磷脂，然而其分子机制和该化合物的绝对构型尚未明确。基于此，该课题拟采用现代绝对构型研究的NMR法，对阿扎霉素F5a进行合理降解，随后测定降解产物的绝对构型，并结合特定片段的模型化合物合成及NMR数据比对，反过来构建和确证阿扎霉素F5a的绝对构型；同时，分别结合以磷脂酰甘油和MRSA细胞膜脂质制备的模拟生物膜，采用拉曼光谱、原子力显微镜、差示扫描量热和NMR等现代波谱和生物物理技术，并借助分子动力学模拟，对阿扎霉素F5a抗MRSA细胞膜磷脂作用的分子机制进行深入研究，为新型抗MRSA药物的研发打下基础。

耐甲氧西林金黄色葡萄球菌（MRSA）对多种结构类型的抗生素相继耐药，新型抗MRSA药物的研发尤为迫切。天然产物阿扎霉素F5a为链霉菌代谢所产36元大环内酯的代表性化合物，申请人2012年首次报道其相对构型后，发现其具有显著的抗MRSA活性，且前期研究预示阿扎霉素F5a可作用于MRSA的细胞膜磷脂，然而其分子机制和该化合物的绝对构型尚未明确。基于此，该课题采用现代绝对构型研究的NMR法和基于偶合常数的构型分析法等，确定了阿扎霉素F5a分子结构中18个手性中心的绝对构型分别为：6S, 7S, 9S, 10S, 11R, 14S, 15S, 17R, 18S, 19R, 21R, 23R, 25S, 27R, 29S, 34R, 35R, 36S。同时，分别制备含有不同阿扎霉素F5a的磷脂酰甘油和MRSA细胞膜脂质制备的模拟生物膜，采用差示扫描量热、ATR-FTIR和31P NMR 等现代波谱和生物物理技术，借助对阿扎霉素F5a分别与磷脂酰甘油、磷脂酰甘油/赖氨酰磷脂酰甘油组成两个系统的分子动力学模拟，并结合膜磷壁酸对阿扎霉素F5a诱导MRSA细胞自溶的保护效果研究，确定了阿扎霉素F5a作用于MRSA细胞膜磷脂的双靶点分子机制，即：阿扎霉素F5a可通过内酯环与细胞膜磷脂的极性头结合、侧链胍基靶向膜磷壁酸引起细胞膜损伤和细胞膜渗透性的改变，从而导致细胞内容物外泄，并最终启动MRSA菌体的自溶，以达到杀菌的效果。从而为新型抗MRSA药物的可能药物研发打下坚实基础，具有潜在的社会效益和经济效益。另外，课题研究中发现的联合用药防细菌耐药新规律可为联合用药抵抗抗菌药物耐药性的理论研究和临床实践打下了坚实基础，在抗菌药物耐药性严重威胁人类公共健康和全球经济的当今，潜在巨大的社会效益和经济效益。