title: Mode of action and biochemical characterization of REP8839, a novel inhibitor of methionyl-tRNA synthetase.
abstract: Aminoacyl-tRNA synthetases have attracted interest as essential and novel targets involved in bacterial protein synthesis. REP8839 is a potent inhibitor of MetS, the methionyl-tRNA synthetase in Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), and in Streptococcus pyogenes. The biochemical activity of REP8839 was shown by specific inhibition of purified S. aureus MetS (50% inhibitory concentration, <1.9 nM). Target specificity was confirmed by overexpression of the metS gene in S. aureus, resulting in an eightfold increase in the MIC for REP8839. Macromolecular synthesis assays in the presence of REP8839 demonstrated a dose-dependent inhibition of protein synthesis and RNA synthesis in S. pneumoniae R6, but only protein synthesis was affected in an isogenic rel mutant deficient in the stringent response. Strains with reduced susceptibility to REP8839 were generated by selection of strains with spontaneous mutations and through serial passages. Point mutations within the metS gene were mapped, leading to a total of 23 different amino acid substitutions within MetS that were located around the modeled active site. The most frequent MetS mutations were I57N, leading to a shift in the MIC from 0.06 microg/ml to 4 microg/ml, and G54S, resulting in a MIC of 32 microg/ml that was associated with a reduced growth rate. The mutation prevention concentration was 32 microg/ml in four S. aureus strains (methicillin-sensitive S. aureus and MRSA), which is well below the drug concentration of 2% (20,000 microg/ml) in a topical formulation. In conclusion, we demonstrate by biochemical, physiologic, and genetic mode-of-action studies that REP8839 exerts its antibacterial activity through specific inhibition of MetS, a novel target.
authors.affiliation: Replidyne, Inc., Louisville, Colorado 80027, USA. uochsner@replidyne.com
authors.forename: Urs A
authors.initials: UA
authors.lastname: Ochsner
authors.forename: Casey L
authors.initials: CL
authors.lastname: Young
authors.forename: Kimberley C
authors.initials: KC
authors.lastname: Stone
authors.forename: Frank B
authors.initials: FB
authors.lastname: Dean
authors.forename: Nebojsa
authors.initials: N
authors.lastname: Janjic
authors.forename: Ian A
authors.initials: IA
authors.lastname: Critchley
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D002874:Chromosome Mapping; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D008712:Methicillin / Q000494:pharmacology; D008718:Methionine-tRNA Ligase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D014176:Protein Biosynthesis; D020836:Protein Structure, Quaternary; D012329:RNA, Bacterial / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D012329:RNA, Bacterial; D008718:Methionine-tRNA Ligase; D008712:Methicillin