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

抗生素耐药性的上升是人类健康的一个主要问题。β-内酰胺类抗生素是最重要的一类抗生素，其耐药性通常是由于其被β-内酰胺酶(BLS)分解而产生的。许多产生BL的细菌对多种药物具有耐药性，可能导致无法治愈的感染。一类临床上重要的抗耐药药物是二氮杂二环辛烷类(DBO)，与β-内酰胺类药物(如阿维巴坦-头孢他啶、释放巴坦-亚胺培南)联合使用以治疗复杂的继发感染。有趣的是，正在开发的新的DBO(如Nacubactam，Zidebactam)显示出对青霉素结合蛋白(PBPs，β-内酰胺的细胞靶标)和BLS的抑制，导致所谓的对β-内酰胺作用的“增强剂”效应，并可能用作“双模式”抗生素。为了抑制BLS和PBPS，DBO抑制剂形成稳定的共价酰基酶复合体。利用多尺度计算机模拟，我们计算了一些BLS的内酰胺酰基酶的形成和分解效率，从而预测单个酶是否对特定的β-内酰胺类抗生素具有耐药性(Chem Comm 2014，50,14736；J Chem Inf Model 2019，59,3365；ACS Catal2020，10,6188)及其对BL抑制剂的敏感性(BioChemical 2018，57,3560)。关键的BLS和PBPS与多个抑制剂的络合物的晶体结构现已可用，实验数据提供了证据，在某些情况下，这些络合物可以缓慢降解以再生活性酶，从而潜在地提供了一条抑制抑制剂耐药性的途径。这一多学科项目旨在解决为什么某些DBO同时抑制BLS和PBPS，而其他DBO仅抑制BLS。基于多尺度模拟的计算分析将用于评估一系列DBO与临床相关的丝氨酸BL(例如KPC-2、OXA-48和变异体)以及来自目标细菌的PBPs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。这些分析的准确性将通过使用一系列动力学方法(联席主管斯宾塞)通过实验测定DBO对特定BLS和PBPS的抑制来验证。BL测试集将包括在地方性耐药地区发现的新变种。该项目将在协作的多学科环境中使用最先进的设施提供尖端技术培训(例如www.bristol.ac.uk/amr/)。对DBO抑制的深入了解将有助于开发具有理想特性的新DBO，这可能有助于新的、有效的细菌感染治疗。

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.