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Protecting our beta-lactam antibiotic assets : structural investigation of beta-lactamases

University of British Columbia

Bacterial diseases have an enormous impact on human health. The most widespread class of human antibacterials is the β-lactams that target the transpeptidase activity of penicillin-binding proteins, which are responsible for cross-linking the peptidoglycan cell-wall. However, bacteria have gained resistance to all major classes of β-lactams. To protect the clinical utility of the β-lactams it is essential to understand the structural basis for this resistance. This thesis aims to better understand the molecular details governing extended-spectrum β-lactamase mediated β-lactam resistance, and to gain insights into inhibition of these emerging resistance factors. Recently, a novel resistance factor known as the New Delhi Metallo-β-Lactamase-1 has been found to confer enteric pathogens such as Escherichia coli and Klebsiella pneumoniae with nearly complete resistance to all β-lactams. The 2.1Å resolution crystal structure of K. pneumoniae holo-NDM-1 revealed an expansive active site, which we propose leads to a broader β-lactam substrate specificity. Furthermore, NDM-1 localizes to the bacterial outer-membrane by sucrose density gradient centrifugation. The structural details underpinning the broad-spectrum resistance of NDM-1 was further investigated by analysis of the protein in complex with hydrolyzed β-lactams as well as bound to the inhibitor L-captopril. An analysis of the NDM-1 active site in these structures reveals key features important for the informed design of novel inhibitors of NDM-1 and other metallo-β-lactamases. The novel diazabicyclooctane (DBO) avibactam inhibits a wider range of serine β-lactamases than has been previously observed with clinical β-lactamase inhibitors. To understand the molecular basis and spectrum of inhibition by avibactam, we provide structural and mechanistic analysis of the compound in complex with important class A and D serine β-lactamases. A kinetic analysis of key active-site mutants for class A β-lactamase CTX-M-15 allows us to propose a validated mechanism for avibactam-mediated β-lactamase inhibition including a unique role for S130, which acts as a general base. We then show that avibactam derivatives retain β-lactamase inhibitory properties but also exhibit considerable antimicrobial activity against clinically relevant bacteria via targeting penicillin-binding proteins. Our results provide evidence that structure-activity relationship studies for the purposes of drug discovery must consider both β-lactamases and penicillin-binding proteins as targets.

Text

2016-07-31

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/56341

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/