新型消除MRSA生物膜天然产物阿扎霉素F5a的C-29位杂合衍生及干预前后的胞外蛋白质组研究

Biofilm was an important mechanism for methicillin-resistant Staphylococcus aureus (MRSA) being noticeable resistant to a variety of structural types of antibiotics, and the research and development of new anti-MRSA drugs eradicating biofilm is particularly urgent. After the anti-MRSA activity of a natural product azalomycin F5a and its synergistic anti-MRSA activity together with trimethylhydroquinone were reported by us, the remarkable activity eradicating MRSA biofilm of azalomycin F5a was discovered while the mechanism is still unknown, and its anti-MRSA activity could be improved by forming Schiff base at C-29 position. Based on these, using azalomycin F5a as an initiator, some derivatives introduced hydroquinone or p-quinone group side chain at C-29 position were designed by modern drug design principle such as hybrid antibiotics and bioisostere, and which were achieved by click chemistry. Next, the anti-MRSA biofilm activities of these azalomycin F5a derivatives were determined. Simultaneously, the extracellular proteomes after/before adding azalomycin F5a into the culture contained MRSA biofilm were researched using gelelectrophoresis, HPLC-MS/MS combined protein database search and subcellular localization, and Western Blot for the in-depth mechanism eradicating biofilm of azalomycin F5a. Hereafter, a solid basis for the research and development of novel and efficient anti-MRSA drugs eradicating biofilm will be established.

生物膜（Biofilm）是耐甲氧西林金黄色葡萄球菌（MRSA）产生多重超强耐药的重要机制，消除生物膜的新型抗MRSA药物研发尤为迫切。申请人在报道天然产物阿扎霉素F5a的抗MRSA活性和协同三甲基氢醌抗MRSA作用后，发现该化合物具有显著的消除MRSA生物膜作用，且C-29位的Schiff碱修饰可显著增强其抗MRSA活性。基于此，该课题以阿扎霉素F5a为起始物，采用杂合抗生素、生物电子等排等现代药物设计思路和Click chemistry高效合成法，在C-29位以Schiff碱的形式引入含对苯酚或对苯醌的侧链，并测定所得衍生物的抗MRSA生物膜作用；同时采用凝胶电泳、HPLC-MS/MS和蛋白质印迹法等现代蛋白质组研究方法和技术，结合蛋白质数据库检索等，对阿扎霉素F5a干预MRSA生物膜前后的胞外蛋白质组进行系统研究，以阐明其消除生物膜的深入机制，为新型高效抗MRSA药物的研发打下基础。

生物膜（Biofilm）是耐甲氧西林金黄色葡萄球菌（MRSA）产生多重超强耐药的重要机制，消除生物膜的新型抗MRSA药物研发尤为迫切。申请人在报道天然产物阿扎霉素F5a的抗金葡球菌活性后，发现该化合物具有显著的消除金葡球菌生物膜作用，且C-29位的修饰可显著增强其抗金葡球菌活性。基于此，该课题一方面采用凝胶电泳、UPLC-MS/MS和PRM验证等现代蛋白质组研究方法和技术，对阿扎霉素F5a干预金葡球菌生长前后的胞外蛋白质组进行研究，以阐明其消除生物膜的机制，共鉴定1935个蛋白，其中上调和下调蛋白分别为222和240个（＞1.3倍），差异蛋白的功能分类、富集和生物信息学显示：阿扎霉素F5a主要作用于细胞膜和膜外成分的合成与代谢等，很多差异蛋白与金葡球菌维持正常的形态和生物膜的消除直接相关，并确定阿扎霉素F5a可快速渗入金葡球菌生物膜内，损伤金葡球菌的细胞膜，引起细胞膜破损或通透性增加，导致部分细菌裂解、自溶和DNase大量释放，从而使对生物膜稳定起重要作用的eDNA的快速降解，并最终导致生物膜的驱散、金葡球菌的释放和被杀灭（即生物膜被消除）。另一方面，课题组采用杂合抗生素的药物设计思路，在阿扎霉素F5a C-29位引入含对苯酚或对苯醌的侧链，研究中基于课题组和其他学者报道约63个类似物和衍生物的归纳与分析，结合分子动力学模拟、药效团模型构建和分子对接，确定末端胍基侧链和内酯环结构是阿扎霉素F5a抗菌活性的必需基团，六元半缩酮环对抗菌活性影响较大，去掉丙二酸单酰基可增强抗菌活性，内酯环上双键氢化、脱甲基或甲基取代对抗菌活性影响较小；基于此，课题组采用骨架跃迁的方法对其进行了分子改造。上述研究结果不仅为该类化合物在生物医药领域的研发打下了坚实基础，还给抗菌和消除细菌生物膜机制、抗细菌毒力因子LTA的机制研究等提供了重要参考，更为结构简单、成药性更强的新型高效抗菌候选药物的研发打下基础。

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