Time resolved structural analyses of β-lactam/β-lactam inhibitor binding to AmpC

SUMMARY Antibiotic resistance is a widespread phenomenon and represents a global threat that is associated with nearly five million deaths a year globally. β-lactams are the most widely used class of antibiotics in treating human infection because of their proven safety and effectiveness. β-lactam resistance refers to the ability of bacteria to withstand the effects of β-lactams. Enzymatic inactivation by β-lactamases is particularly relevant to β- lactam antibiotics as these enzymes hydrolyze the β-lactam ring, thereby rendering them ineffective. AmpC β- lactamases (AmpCs) are produced by most gram-negative bacteria that infect humans including Pseudomonas aeruginosa, and Acinetobacter baumannii, and many species in the order Enterobacterales. They confer resistance to a wide range of β-lactams commonly used to treat bacterial infections and are key drivers of antimicrobial resistance in these bacteria. In general, AmpCs hydrolyze penicillins, cephalosporins and monobactams, and are not inhibited by classic β-lactamase inhibitors (BLIs) but do not confer resistance to carbapenems and to newer β-lactam/BLI combinations such as ceftazidime-avibactam. Extended-spectrum AmpCs display an increased catalytic efficiency toward extended-spectrum cephalosporins and, in some cases, imipenem, the prototypical carbapenem. Extended-spectrum AmpCs may also be less susceptible to inhibition by BLIs. Despite a plethora of studies of AmpC biology, there are several major knowledge gaps. Specifically, all crystal structures of AmpC s in complex with β-lactams or BLIs capture the acyl-enzyme complex, and we lack key insight into the initial binding interactions, intermediate states and kinetic mechanisms associated with the acylation and deacylation steps of the reaction. This knowledge gap significantly limits our understanding of how mutations in extended-spectrum AmpCs confer increased catalytic efficiency toward cephalosporins and/or decrease the inhibitory activity of BLIs. Our central hypothesis is that the application of time-resolved crystallography, coupled with biochemical and microbiological approaches, will provide unprecedented insight into AmpC structure, catalysis and substrate specificity. In Specific Aim 1, we will elucidate the binding, intermediate states and hydrolysis of β-lactam antibiotics by wild type (WT) and extended-spectrum AmpCs. In Specific Aim 2 we will elucidate the reaction mechanisms for BLIs for WT and extended-spectrum AmpCs. The data derived from these Aims will provide a comprehensive understanding for how WT and extended-spectrum AmpC interact with, and hydrolyze, β-lactams and BLIs. This critical insight will ultimately pave the way for developing more effective β-lactam antibiotics and BLIs, which is crucial in the ongoing battle against antibiotic-resistant gram-negative bacteria. Project Number: 1R01AI197205-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Guillermo Calero (+1 co-PI) | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $799,572 | Activity Code: R01 | Study Section: Macromolecular Structure and Function A Study Section[MSFA] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19720501