title: Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase (TilS) by High-Throughput Screening.
abstract: A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series.
authors.affiliation: Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, USA adam.shapiro@astrazeneca.com.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.affiliation: Screening Sciences, Discovery Sciences, AstraZeneca Pharmaceuticals, Macclesfield, Cheshire, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Plant
authors.affiliation: Screening Sciences, Discovery Sciences, AstraZeneca Pharmaceuticals, Macclesfield, Cheshire, UK.
authors.forename: Jarrod
authors.initials: J
authors.lastname: Walsh
authors.affiliation: Chemistry Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Sylvester
authors.affiliation: Structure & Biophysics, Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Hu
authors.affiliation: Reagents & Assay Development, Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.affiliation: Reagents & Assay Development, Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA, USA.
authors.forename: Stephania
authors.initials: S
authors.lastname: Livchak
authors.affiliation: Chemistry Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, USA.
authors.forename: Sharon
authors.initials: S
authors.lastname: Tentarelli
authors.affiliation: Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Thresher
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000926:Anticodon; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D008239:Lysine / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013329:Structure-Activity Relationship
chemical_list: D000926:Anticodon; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; C478460:tilS protein, E coli; D008239:Lysine
keywords: TilS; inhibitor; lysidine; tRNAIle lysidine synthetase; ultrahigh-throughput screening