Countering antimicrobial resistance: investigating activity of beta-lactamase inhibitors using atomistic simulation and experiment

不断上升的抗生素耐药性是人类健康的一个主要问题。对β-内酰胺类抗生素（最重要的一类抗生素）的耐药性通常是通过β-内酰胺酶（BL）分解而产生的。许多产生BL的细菌具有多重耐药性，可能导致无法治疗的感染。对抗这种耐药性的一类临床上重要的BL抑制剂是二氮杂双环辛烷（DBO），与β-内酰胺类（例如阿维巴坦-头孢他啶、阿维巴坦-亚胺培南）一起用于治疗复杂的二级护理感染。有趣的是，开发中的新DBO（例如纳库巴坦、齐德巴坦）显示出对青霉素结合蛋白（PBPs，β-内酰胺的细胞靶点）以及BL的抑制，导致对β-内酰胺作用的所谓“增强剂”效应，并可能用作“双模式”抗生素。为了抑制BL和PBP，DBO抑制剂形成稳定的共价酰基-酶复合物。使用多尺度计算机模拟，我们计算了许多BL的β-内酰胺酰基酶形成和分解的效率，从而预测单个酶是否可以赋予对特定β-内酰胺的抗性。（Chem Comm 2014，50，14736; J Chem Inf Model 2019，59，3365; ACS Catal 2020，10，6188）及其对BL抑制剂的敏感性（Biochemistry 2018，57，3560）。关键的BLs和PBPs与多种抑制剂的复合物的晶体结构现在是可用的，实验数据提供证据表明，在某些情况下，这些复合物可以缓慢降解，以再生活性酶，可能提供一个途径抑制剂resistance.This多学科项目旨在解决为什么某些DBO抑制BLs和PBPs，而其他人只抑制BLs。基于多尺度模拟的计算试验将用于评估由一系列DBO与临床相关丝氨酸BL（例如KPC-2、OXA-48和变体）以及来自靶细菌的PBP形成的酰基酶的形成和分解。这是具有挑战性的，因为需要探索不同的反应机制。将使用一系列动力学方法（共同监督人Spencer），通过实验测定特定BL和PBPs的DBO抑制来验证这些测定的准确度。BL测试集将包括在地方性抗药性地区发现的新变种，该项目将在多学科协作环境中利用最先进的设施提供尖端技术培训（例如www.bristol.ac.uk/amr/）。对DBO抑制的深入了解将为开发具有理想特性的新DBO提供信息，这可能有助于细菌感染的新的有效治疗。

Rising antibiotic resistance is a major problem for human health. Resistance to beta-lactams, the single most important antibiotic class, often arises through their breakdown by beta-lactamases (BLs). Many BL producing bacteria are multi-drug resistant and may cause untreatable infections. A clinically important class of BL inhibitors to combat this resistance are diazabicyclooctanes (DBOs), used together with beta-lactams (e.g. avibactam-ceftazidime, relebactam-imipenem) to treat complicated secondary care infections. Interestingly, new DBOs in development (e.g. Nacubactam, Zidebactam) show inhibition of penicillin binding proteins (PBPs, the cellular targets of the -lactams) as well as BLs, leading to a so-called "enhancer" effect upon beta-lactam action and possible use as 'dual mode' antibiotics. To inhibit BLs and PBPs, DBO inhibitors form stable covalent acyl-enzyme complexes. Using multi-scale computer simulations, we have calculated the efficiency of -lactam acyl-enzyme formation and breakdown for a number of BLs, thereby predicting whether individual enzymes can confer resistance to specific beta-lactams (Chem Comm 2014, 50, 14736; J Chem Inf Model 2019, 59, 3365; ACS Catal 2020, 10, 6188) and their susceptibility to BL inhibitors (Biochemistry 2018, 57, 3560). Crystal structures of complexes of key BLs and PBPs with multiple inhibitors are now available and experimental data provide evidence that in some cases these complexes can slowly degrade to regenerate active enzyme, potentially providing a route to inhibitor resistance.This multidisciplinary project aims to address why certain DBOs inhibit both BLs and PBPs, whereas others inhibit BLs only. Computational assays based on multi-scale simulations will be used to assess formation and breakdown of the acyl-enzymes formed by a range of DBOs with clinically relevant serine BLs (e.g. KPC-2, OXA-48 and variants) as well as PBPs from target bacteria. This is challenging, as different reaction mechanisms will need to be explored. The accuracy of these assays will be validated by experimental determination of DBO inhibition of specific BLs and PBPs, using a range of kinetic methods (co-supervisor Spencer). The BL test set will include new variants identified in regions with endemic resistance.The project will provide training in cutting-edge techniques using state-of-the-art facilities in the context of a collaborative multi-disciplinary environment (e.g. www.bristol.ac.uk/amr/). Insights obtained into DBO inhibition will inform development of new DBOs with desirable characteristics, which may contribute to new, effective treatment of bacterial infections.