β-内酰胺酶MβLs和SβLs的双重抑制及在活菌体内的实时监测

β-内酰胺酶包括金属β-内酰胺酶（MβLs）和丝氨酸β-内酰胺酶（SβLs）介导细菌之耐药性的遏制是G20峰会（2016）公布的全球行动计划和中国《遏制细菌耐药国家行动计划》。该项目拟以大肠杆菌表达并纯化细菌耐药靶酶MβLs和SβLs；构建阿维巴坦-唑基硫代乙酰胺/罗丹宁衍生物，表征其结构，评价其对两类酶的抑制活性和抑制类型；细菌非侵入性UV-Vis及ITC实时监测其对活菌体内靶酶的真实抑制活性，筛选可用于临床目的的酶抑制剂；荧光共聚焦显微镜(3D-SIM)实时监测其在活菌体内的摄入及与其它胞内成分的互动等；MIC评价其对多重耐药细菌的遏制效能；NMR表征其与靶酶在液/动态的键合，MS评价其与靶酶作用的强度，分子对接及定点突变阐明其与靶酶的作用模式；悬滴/浸泡法制备抑制剂-MβLs/SβLs复合物的有序晶体，解析结构获得其作用模式的直接证据，为发展新型抗耐药菌药物提供有重要价值的信息。

The bacterial resistance mediated by β-lactamases including metallo-β-lactamase (MβLs) and serine-β-lactamase (SβLs) is the global action plan published at the G20 summit (2016) and the china national action plan to curb bacterial resistance. The project aims to express and purify the bacterial resistant target enzyme MβLs and SβLs in E. coli; construct Avibactam-azolethioacetamide/rhodanine derivatives, characterize their structure, evaluate their dual inhibitory activity and inhibitory type; real-time monitor their real activity against the target enzymes in living bacteria by bacterial non-invasive UV-Vis and ITC methods, and screen enzyme inhibitors for clinical purposes; monitor their intake in living bacteria and interaction with other intracellular components by fluorescence confocal microscopy (3D-SIM); MIC evaluate their efficacy against bacterial multidrug resistance; NMR characterize their binding with the target enzymes in liquid/dynamic; MS evaluate their intensity of action with the target enzymes; molecule docking and site-directed mutation to elucidate their mode of action with the target enzymes; preparation of ordered crystals of inhibitor-MβLs/SβLs complexes by hanging drop/soaking method, resolve the structure to obtain te direct evidence of their action mode, and provide important information for the development of new anti-resistant bacteria drugs.