β-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli, but their use is increasingly compromised by β-lactamases, including the chromosomally encoded class C AmpC serine-β-lactamases (SBLs). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimize the DBO core. We report kinetic and structural studies, including four high-resolution crystal structures, concerning inhibition of the AmpC serine-β-lactamase from E. coli (AmpC_EC) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpC_EC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (apparent inhibition constant [K_iapp], 0.69 μM) against AmpC_EC compared to that of the other DBOs (K_iapp = 5.0 to 7.4 μM) due to an ∼10-fold accelerated carbamoylation rate. However, zidebactam also has an accelerated off-rate, and with sufficient preincubation time, all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpC_EC-zidebactam carbamoyl complex compared to those for the other DBOs. The results suggest the carbamoyl complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.

中文翻译：

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二氮杂双环辛烷抑制大肠杆菌AmpC {beta}-内酰胺酶的结构研究

β-内酰胺类抗生素目前是致病性大肠杆菌感染最重要的治疗方法，但它们的使用越来越受到 β-内酰胺酶的影响，包括染色体编码的 C 类 AmpC 丝氨酸-β-内酰胺酶 (SBL)。二氮杂双环辛烷 (DBO) avibactam 是一种有效的 AmpC 抑制剂；avibactam 联合头孢他啶的临床成功激发了优化 DBO 核心的努力。我们报告了动力学和结构研究，包括四种高分辨率晶体结构，涉及抑制大肠杆菌中的 AmpC 丝氨酸-β-内酰胺酶(AmpC _EC) 通过临床相关的基于 DBO 的抑制剂：avibactam、relebactam、nacubactam 和 zidebactam。动力学分析和基于质谱的测定用于研究它们的 AmpC _EC抑制机制。结果表明，在我们的测定条件下，与其他 DBO（ K _iapp = 5.0 至 7.4 μM）相比，齐地巴坦对 AmpC _EC的效力增加（表观抑制常数 [ K _iapp ]，0.69 μM） ，这是由于~10 -倍加速的氨甲酰化速率。然而，齐地巴坦也具有加速解离率，并且在足够的预孵育时间下，所有 DBO 都表现出相似的效力。晶体学分析表明 AmpC _{EC具有更大的构象自由度}-zidebactam 氨基甲酰基复合物与其他 DBO 相比。结果表明，氨基甲酰基复合物的寿命应该是开发用于临床重要的 C 类 SBL 的基于 DBO 的 SBL 抑制剂的考虑因素。