title: Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay.
abstract: A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 microg/mL). The results show that mass spectrometry-based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.
authors.affiliation: Department of Antimicrobial Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
authors.forename: Erik F
authors.initials: EF
authors.lastname: Langsdorf
authors.forename: Asra
authors.initials: A
authors.lastname: Malikzay
authors.forename: William A
authors.initials: WA
authors.lastname: Lamarr
authors.forename: Dayna
authors.initials: D
authors.lastname: Daubaras
authors.forename: Cynthia
authors.initials: C
authors.lastname: Kravec
authors.forename: Rumin
authors.initials: R
authors.lastname: Zhang
authors.forename: Richard
authors.initials: R
authors.lastname: Hart
authors.forename: Frederick
authors.initials: F
authors.lastname: Monsma
authors.forename: Todd
authors.initials: T
authors.lastname: Black
authors.forename: Can C
authors.initials: CC
authors.lastname: Ozbal
authors.forename: Lynn
authors.initials: L
authors.lastname: Miesel
authors.forename: Charles A
authors.initials: CA
authors.lastname: Lunn
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors*; D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008826:Microbial Sensitivity Tests; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase; D004121:Dimethyl Sulfoxide