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

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.