Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with β-Lactones

Abstract

β-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to β-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of β-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four β-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein–ligand interactions and characterized the β-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

Drug Discovery in the Field of β-Lactams: An Academic Perspective

“…Among these compounds, 24 showed potent activity against the multi-drug-resistant (MDR) S. aureus NRS70 strain (MIC values of 3.13 µg/mL). (21 [211], 22 [196], 23 [212], LYS228 [213], 24 [217], 25 [218], 26 [219], 27 [152], 28 [220], ETX0462 [221], 29 [222], 30 [223]).…”

“…Compound 25 consists of a dihydroxypyridone siderophore conjugated to a modified aminothiazoylglycyl cephalosporin. This modification allows the drug to be (21 [211], 22 [196], 23 [212], LYS228 [213], 24 [217], 25 [218], 26 [219], 27 [152], 28 [220], ETX0462 [221], 29 [222], 30 [223]).…”

“…These compounds have been used as chemical probes to selectively target specific PBPs in S. pneumoniae [225,226]. A successful example was compound 29, which was shown to bind to PBP2a in silico [222]. Further insights into interactions between β-lactones and PBP1b were provided by crystallography and additional molecular docking studies.…”

β-Lactams from the Ocean

“…These rings have commonalities beyond ring size. β-Lactone structures are successful affinity probes of the penicillin-binding proteins (PBPs) [ 133 , 134 , 135 ]. The product of Class D β-lactamase hydrolysis of carbapenems is a β-lactone [ 136 , 137 ].…”

Penicillin-binding protein (PBP) inhibitor development: A 10-year chemical perspective

“…Structural biology has been extensively employed to characterize complexes between PBPs and β-lactam analogs with the objective of understanding catalytic details, resistance mechanisms, and developing novel ligands. 8,[20][21][22][23][24][25][26][27][28] In these structures, the nucleophilic serine is covalently associated to the β-lactam carbonyl as an ester. Despite these efforts, until recently no reports had been published relating complex formation between a PBP and a ligand product resulting from hydrolysis by a β-lactamase.…”