Spectrum of activity and mode of action of REP3123, a new antibiotic to treat Clostridium difficile infections.
OBJECTIVE
The aim of this study was to characterize the antimicrobial profile of REP3123, a novel inhibitor of methionyl-tRNA synthetase (MetRS) in development for the treatment of Clostridium difficile infection.


METHODS
The spectrum of activity of REP3123 was determined by susceptibility testing of C. difficile and non-target organisms. The mode of action was studied by enzyme inhibition assays, macromolecular synthesis assays, target overexpression and selection of spontaneous resistant mutants.


RESULTS
REP3123 was active against a collection of 108 clinical isolates of C. difficile and against epidemic, moxifloxacin-resistant BI/NAP1/027 strains (MIC range=0.5-1 mg/L and MIC(90) = 1 mg/L). The spectrum of activity included clinically important aerobic Gram-positive cocci such as Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis and Enterococcus faecium (MIC(90)s < 1 mg/L), but REP3123 was not active against most Gram-negative bacteria. REP3123 targeted C. difficile MetRS with a calculated inhibition constant (K(i)) of 0.020 nM, and selectivity was >1000-fold over human mitochondrial and cytoplasmic MetRS. The specific mode of action within bacterial cells was demonstrated by macromolecular synthesis assays that showed inhibition of protein synthesis by REP3123, and by metS overexpression, which resulted in a 16-fold increase in MIC for REP3123. Spontaneous REP3123-resistant mutants of C. difficile (MICs, 4-128 mg/L) arose with frequencies of 10(-8)-10(-9) and harboured distinct point mutations within the metS gene, resulting in 13 different amino acid substitutions. Most of the MetRS substitutions caused reduced catalytic efficiency and a growth fitness burden.


CONCLUSIONS
REP3123 demonstrated a favourable microbiological profile and was found to target C. difficile with high specificity and selectivity.
Critchley
Ian A
IA
Replidyne, Inc., Louisville, CO 80027, USA.
Green
Louis S
LS
Young
Casey L
CL
Bullard
James M
JM
Evans
Ron J
RJ
Price
Melissa
M
Jarvis
Thale C
TC
Guiles
Joseph W
JW
Janjic
Nebojsa
N
Ochsner
Urs A
UA
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D001578:Benzopyrans / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018628:Gene Dosage; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects*; D006801:Humans; D008718:Methionine-tRNA Ligase / Q000037:antagonists & inhibitors* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D017354:Point Mutation; D014176:Protein Biosynthesis / Q000187:drug effects; D013876:Thiophenes / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001578:Benzopyrans; D004791:Enzyme Inhibitors; C541771:REP 3123; D013876:Thiophenes; D008718:Methionine-tRNA Ligase