title: An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis.
abstract: The lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LPS biosynthesis, leading to a truncated LPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis.
authors.affiliation: Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
authors.forename: Gladys P
authors.initials: GP
authors.lastname: De Leon
authors.forename: Nadine H
authors.initials: NH
authors.lastname: Elowe
authors.forename: Kalinka P
authors.initials: KP
authors.lastname: Koteva
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
authors.forename: Gerard D
authors.initials: GD
authors.lastname: Wright
mesh_terms: D000247:Adenosine Diphosphate Sugars / Q000096:biosynthesis*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D016695:Glycosyltransferases / Q000037:antagonists & inhibitors*; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008826:Microbial Sensitivity Tests; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors
chemical_list: D000247:Adenosine Diphosphate Sugars; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D016695:Glycosyltransferases; C086735:heptosyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); D009713:Nucleotidyltransferases; C445302:D,D heptose 7-phosphate kinase-D,D-heptose 1-phosphate adenylyltransferase, E coli