title: A high-throughput-compatible fluorescence anisotropy-based assay for competitive inhibitors of Escherichia coli UDP-N-acetylglucosamine acyltransferase (LpxA).
abstract: LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.
authors.affiliation: AstraZeneca R&D Boston, Waltham, MA 02451, USA. adam.shapiro@astrazeneca.com
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.forename: Philip L
authors.initials: PL
authors.lastname: Ross
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.forename: Stephania
authors.initials: S
authors.lastname: Livchak
authors.forename: Gunther
authors.initials: G
authors.lastname: Kern
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Beth
authors.initials: B
authors.lastname: Andrews
authors.forename: Jason
authors.initials: J
authors.lastname: Thresher
mesh_terms: D000213:Acyl Carrier Protein / Q000378:metabolism; D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001667:Binding, Competitive; D020134:Catalytic Domain; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D020128:Inhibitory Concentration 50; D008024:Ligands; D008050:Lipid A / Q000378:metabolism; D009227:Myristic Acids / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D004791:Enzyme Inhibitors; D008024:Ligands; D008050:Lipid A; D009227:Myristic Acids; D010455:Peptides; C052121:UDP-3-O-(3-hydroxymyristoyl)-N-acetylglucosamine; D014537:Uridine Diphosphate N-Acetylglucosamine; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase