Targeting Bacterial Cell Wall Peptidoglycan Synthesis by Inhibition of Glycosyltransferase Activity

Abstract

Synthesis of bacterial cell wall peptidoglycan requires glycosyltransferase enzymes that transfer the disaccharide-peptide from lipid II onto the growing glycan chain. The polymerization of the glycan chain precedes cross-linking by penicillin-binding proteins and is essential for growth for key bacterial pathogens. As such, bacterial cell wall glycosyltransferases are an attractive target for antibiotic drug discovery. However, significant challenges to the development of inhibitors for these targets include the development of suitable assays and chemical matter that is suited to the nature of the binding site. We developed glycosyltransferase enzymatic activity and binding assays using the natural products moenomycin and vancomycin as model inhibitors. In addition, we designed a library of disaccharide compounds based on the minimum moenomycin fragment with peptidoglycan glycosyltransferase inhibitory activity and based on a more drug-like and synthetically versatile disaccharide building block. A subset of these disaccharide compounds bound and inhibited the glycosyltransferase enzymes, and these compounds could serve as chemical entry points for antibiotic development.

Network-Based Metabolism-Centered Screening of Potential Drug Targets in Klebsiella pneumoniae at Genome Scale

“…Penicillin-binding proteins (PBPs) carrying transpeptidase and/or glycosyltransferase activities are involved in the final stages of peptidoglycan synthesis. Polymerization of peptidyl disaccharide subunits is managed through the glycosyltransferase activity, and cross-linking by peptide bridges is catalyzed through the transpeptidase activity (Derouaux et al, 2013;Mesleh et al, 2016). Inhibition of the peptidoglycan synthesis was reported to result in bacterial cell lysis and subsequently death of the cell (Derouaux et al, 2013).…”

Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials

“…These labeled substrates facilitated the development of activity assays [ 3 , 143 , 155 , 156 , 157 ]. The availability of substrate variants that serve as donor only, acceptor only, or that are not processed by the GT enzyme and thus function as inhibitors (see below) contributed greatly to the understanding of the specificity of the GT and TP catalyzed reactions [ 138 , 147 , 158 , 159 , 160 , 161 , 162 , 163 ]. Thanks to these substrates, lasting problems have been solved.…”

“…Mesleh et al [ 159 ] performed computational solvent mapping of a GT to identify druggable pockets in the active site and to help in the design of new compounds. The major hotspot regions were identified in the donor site, overlapping with binding sites of rings EFG of moenomycin A near the essential glutamate and other conserved residues of the active site, confirming that the EFG pharmacophore is the best lead for GT inhibitor design.…”

“…During this assay, the polymerase activity of GT is accompanied by a fluorescence decrease when dansyl lipid II is converted to a polymer followed by muramidase hydrolysis. This assay has been further optimized and adapted to microtiter plate format [ 141 , 159 ]. It can be used for the screening of compounds but the presence of fluorescent molecules often interferes with the assay.…”

“…The LC step can be time-consuming and thus may not be suitable for HTS. The second report [ 159 ] describes a new GT activity assay developed using the RapidFire High-Throughput MS solid phase extraction system (Agilent Technologies). It is based on the detection of the C55PP product released during the reaction.…”