High-throughput screening identifies novel inhibitors of the acetyltransferase activity of Escherichia coli GlmU.
The bifunctional GlmU protein catalyzes the formation of UDP-N-acetylglucosamine in a two-step reaction using the substrates glucosamine-1-phosphate, acetyl coenzyme A, and UTP. This metabolite is a common precursor to the synthesis of bacterial cell surface carbohydrate polymers, such as peptidoglycan, lipopolysaccharide, and wall teichoic acid that are involved in the maintenance of cell shape, permeability, and virulence. The C-terminal acetyltransferase domain of GlmU exhibits structural and mechanistic features unique to bacterial UDP-N-acetylglucosamine synthases, making it an excellent target for antibacterial design. In the work described here, we have developed an absorbance-based assay to screen diverse chemical libraries in high throughput for inhibitors to the acetyltransferase reaction of Escherichia coli GlmU. The primary screen of 50,000 drug-like small molecules identified 63 hits, 37 of which were specific to acetyltransferase activity of GlmU. Secondary screening and mode-of-inhibition studies identified potent inhibitors where compound binding within the acetyltransferase active site was requisite on the presence of glucosamine-1-phosphate and were competitive with the substrate acetyl coenzyme A. These molecules may represent novel chemical scaffolds for future antimicrobial drug discovery. In addition, this work outlines the utility of catalytic variants in targeting specific activities of bifunctional enzymes in high-throughput screens.
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.
Mark P
MP
Pereira
Jan E
JE
Blanchard
Cecilia
C
Murphy
Steven L
SL
Roderick
Eric D
ED
Brown
D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D015195:Drug Design; D004926:Escherichia coli / Q000187:drug effects*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D002800:Cholinesterase Inhibitors; D029968:Escherichia coli Proteins; C520333:GlmU protein, E coli; D009097:Multienzyme Complexes; D014537:Uridine Diphosphate N-Acetylglucosamine