微生物来源的细菌烯脂酰ACP还原酶抑制剂的新型细胞筛选模型的建立与应用研究

To overcome the emerging drug resistance,the discovery of novel antibiotic chemical scaffolds with new modes of action is crucial strategy for saving lives. Bacterial enoyl-ACP reductase as an effective drug screening targets have been widely used for screening of new antimicrobial agents. Enoyl ACP reductase displays diversity among pathogenic bacteria, but the screening of antimicrobial drug only targets a single enoyl ACP reductase, so antimicrobial drug shows narrow antibacterial spectrum and need to create different screening system for different enoyl-ACP reductase. These reasons restrain the velocity of screening. Therefore, to contrapose the theoretical research base that based on diversity in bacterial enoyl ACP reductase, the difference between the enoyl ACP reductase isozymes and can substitute for each other as well as FabI is the only target of triclosan in E. coli, the project uses molecular biology techniques to build novel cell-based screening model for inhibitors which targets to three enoyl acyl ACP reductase FabI,FabK and FabV,and using this model to perform the antibacterial substances screening validation and research,in order to achieve industrialization application,and to lay a foundation for the development of new antibiotics with independent intellectual property rights.

寻找具有新作用方式的抗菌药物是防治不断出现的耐药性致病细菌感染的重要策略。细菌烯脂酰ACP还原酶作为有效的药物筛选靶点已被广泛用于新抗菌药物的筛选。但是由于烯脂酰ACP还原酶在致病细菌中存在多样性，而当前抗菌药物的筛选只针对单一的靶点，致使筛到的抗菌药物的抗菌谱较窄且针对不同烯脂酰ACP还原酶靶点需要建立不同的筛选体系，制约了抗菌药物筛选的速度。因此，本项目针对细菌烯脂酰ACP还原酶多样性，基于烯脂酰ACP还原酶的同工酶之间的差异性及可相互替代性以及FabI是三氯生在大肠杆菌中的唯一作用靶点的理论研究基础，利用分子生物学技术构建以三种烯脂酰ACP还原酶FabI、FabK和FabV为靶点的新型烯脂酰ACP还原酶抑制剂细胞筛选模型，并利用该模型进行抑菌活性物质的筛选验证和研究，以期能进行产业化运用，为具有自主知识产权的新型抗生素的开发奠定基础。

寻找具有新作用方式的抗菌药物是防治不断出现的耐药性致病细菌感染的重要策略。细菌烯脂酰ACP还原酶作为有效药物筛选靶点已被广泛用于新抗菌药物的筛选。但是由于烯脂酰ACP还原酶在致病细菌中存在多样性，而当前抗菌药物的筛选只针对单一的靶点，致使筛到的抗菌药物的抗菌谱较窄且针对不同烯脂酰ACP还原酶靶点需要建立不同的筛选体系，制约了抗菌药物筛选的速度。因此，本项目针对细菌烯脂酰ACP还原酶多样性，基于烯脂酰ACP还原酶的同工酶之间的差异性及可相互替代性以及FabI是三氯生在大肠杆菌中的唯一作用靶点的理论研究基础，利用分子生物学技术构建了以三种烯脂酰ACP还原酶FabI、FabK和FabV为靶点的高效而灵敏的烯脂酰ACP还原酶抑制剂细胞筛选模型，并利用该模型进行抑菌活性物质的筛选验证和研究。从8186株放线菌中筛选得到33株阳性菌株，筛选阳性率为0.4%，利用所纯化的细菌烯脂酰ACP还原酶酶活测定体系, 验证了所建立的细胞水平筛选模型具有较高的准确性和特异性。研究结果表明其中一个阳性样品在细胞水平和蛋白水平均具有较好的活性，值得进行深入的研究。这些工作为具有自主知识产权的新型抗生素的开发奠定了基础。