Antibacterial activity of REP8839, a new antibiotic for topical use.

REP8839 is a novel methionyl-tRNA synthetase (MetS) inhibitor with potent antibacterial activity against clinical isolates of Staphylococcus aureus, Streptococcus pyogenes, and other clinically important gram-positive bacteria but little activity against gram-negative bacteria. All isolates of S. aureus, including strains resistant to methicillin, mupirocin, vancomycin, and linezolid were susceptible to REP8839 at concentrations of < or =0.5 microg/ml. REP8839 was also active against Staphylococcus epidermidis, including multiply resistant strains (MIC, < or =0.25 microg/ml). All S. pyogenes isolates were susceptible to REP8839 at concentrations of < or =0.25 microg/ml, suggesting that MetS2, a second enzyme previously identified in Streptococcus pneumoniae, was not present in this organism. REP8839 was highly bound to the protein of human serum, and activity was not greatly influenced by inoculum size but was affected by pH, exhibiting optimal antibacterial activity in a neutral medium rather than a weak acidic medium. Like mupirocin, REP8839 exhibited bacteriostatic activity against key pathogens. The emergence of mupirocin resistance in S. aureus highlights the need for a new topical antibiotic with the ability to inhibit high-level mupirocin-resistant strains and other emerging phenotypes, such as vancomycin-resistant and community-acquired methicillin-resistant isolates.

Mode of action and biochemical characterization of REP8839, a novel inhibitor of methionyl-tRNA synthetase.

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.

Abnormal proteins enhance stress-induced cell death.

The effect of abnormal proteins on cell viability was studied using artificially cleaved polypeptides. Escherichia coli methionyl-tRNA synthetase (MetRS) consists of two distinct domains and its activity is essential for cell viability. The polypeptide chain was split by linker insertion and expressed as two fragments. Two pairs of polypeptides, one split within the N-terminal domain and another at the junction of the two domains retained aminoacylation activity. The in vitro activities of these split mutants were enhanced by the presence of chaperonin, GroESL. However, cells containing these split polypeptides became sensitive to conditions that induce GroESL. The results of this work suggest that an abnormally generated protein can cause cell death under stressful conditions.