Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

Abstract

Penicillin-binding proteins (PBPs) make up an essential class of bacterial enzymes that carry out the final steps of peptidoglycan synthesis and regulate the recycling of this polymeric structure. PBPs are an excellent drug target and have been the most clinically relevant antibacterial target since the 1940s with the introduction of β-lactams. Despite this, a large gap in knowledge remains regarding the individual function and regulation of each PBP homologue in most bacteria. This can be attributed to a lack of chemical tools and methods that enable the study of individual PBPs in an activity-dependent manner and in their native environment. The development of such methods in Gram-negative bacteria has been particularly challenging due to the presence of an outer membrane and numerous resistance mechanisms. To address this, we have developed an optimized live-cell assay for screening inhibitors of the PBPs in Escherichia coli MG1655. We utilized EDTA to permeabilize Gram-negative cells, enabling increased penetration of our readout probe, Bocillin-FL, and subsequent analysis of PBP-inhibition profiles. To identify scaffolds for future development of PBP-selective activity-based probes, we screened ten β-lactams, one diazabicyclooctane, and one monobactam for their PBP-selectivity profiles in E. coli MG1655. These results demonstrate the utility of our assay for the screening of inhibitors in live, non-hypersusceptible Gram-negative organisms.

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

“…Related PBP-occupancy experiments have been carried out for various β-lactams in other bacteria [176,177]. Novel assays have also been developed to simultaneously measure the outer membrane permeability of various β-lactams against carbapenem-resistant K. pneumoniae, Enterobacter cloacae, E. coli, and P. aeruginosa [178][179][180][181], which offers important insights into the interactions between β-lactams and PBPs in situ. Furthermore, there has been increasing interest in improving treatment efficacy by targeting multiple PBPs and potentially some β-lactamases as well, by combining several β-lactams [182,183].…”

kinact/KIValue Determination for Penicillin-Binding Proteins in Live Cells

“…3 Inhibitor potency is commonly characterized by an IC50 value, or the inhibitor concentration required for 50% enzyme inhibition at incubation time t. [4][5][6][7][8][9] Previous work in our lab has explored the use of β-lactams to define IC50 values for the PBP homologs in multiple Gram-positive and Gram-negative organisms. [10][11][12][13][14] These studies aimed to identify inhibitors that would serve as scaffolds to develop activity-based probes (ABPs) selective for individual PBP homologs. While useful for our screen of relative PBP selectivity, the time and substrate concentration dependencies of IC50 values for irreversible inhibitors prohibit absolute inhibitor potency characterization for the study of structure-activity relationships (SAR).…”