ABSTRACT
Cadazolid is a new oxazolidinone-type antibiotic currently in clinical development for the treatment of Clostridium difficile-associated diarrhea. Here, we report investigations on the mode of action and the propensity for spontaneous resistance development in C. difficile strains. Macromolecular labeling experiments indicated that cadazolid acts as a potent inhibitor of protein synthesis, while inhibition of DNA synthesis was also observed, albeit only at substantially higher concentrations of the drug. Strong inhibition of protein synthesis was also obtained in strains resistant to linezolid, in agreement with low MICs against such strains. Inhibition of protein synthesis was confirmed in coupled transcription/translation assays using extracts from different C. difficile strains, including strains resistant to linezolid, while inhibitory effects in DNA topoisomerase assays were weak or not detectable under the assay conditions. Spontaneous resistance frequencies of cadazolid were low in all strains tested (generally <10(-10) at 2× to 4× the MIC), and in multiple-passage experiments (up to 13 passages) MICs did not significantly increase. Furthermore, no cross-resistance was observed, as cadazolid retained potent activity against strains resistant or nonsusceptible to linezolid, fluoroquinolones, and the new antibiotic fidaxomicin. In conclusion, the data presented here indicate that cadazolid acts primarily by inhibition of protein synthesis, with weak inhibition of DNA synthesis as a potential second mode of action, and suggest a low potential for spontaneous resistance development.
Subject(s)
Anti-Bacterial Agents/pharmacology , Clostridioides difficile/drug effects , Drug Resistance, Bacterial/genetics , Protein Biosynthesis/drug effects , Acetamides/pharmacology , Aminoglycosides/pharmacology , Clostridioides difficile/genetics , Clostridioides difficile/growth & development , Clostridioides difficile/metabolism , DNA Gyrase/genetics , DNA Gyrase/metabolism , Drug Resistance, Bacterial/drug effects , Fidaxomicin , Fluoroquinolones/pharmacology , Linezolid , Microbial Sensitivity Tests , Oxazolidinones/pharmacology , Protein Biosynthesis/genetics , RNA/antagonists & inhibitors , RNA/biosynthesis , Recombinant Proteins , Subcellular Fractions/chemistry , Subcellular Fractions/metabolism , Transcription, Genetic/drug effects , Vancomycin/pharmacologyABSTRACT
Aminoacyl-tRNA synthetases (AARSs) are a structurally heterogeneous family of enzymes present in prokaryotes, archaea and eukaryotes. They catalyze the attachment of tRNA to its corresponding amino acid via an aminoacyl adenylate intermediate. Errors in protein synthesis will occur if an incorrect amino acid is attached to the tRNA. To prevent such errors, AARSs have evolved editing mechanisms that eliminate incorrect aminoacyl adenylates (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Various AARSs are the targets of natural antibiotics and are considered validated targets for chemotherapy. We have developed a high-throughput screening (HTS) assay measuring the pre-transfer editing activity of pathogen-derived asparaginyl-tRNA synthetase (AsnRS). This was achieved by monitoring the formation of pyrophosphate via cleavage to phosphate, which was quantified by reaction with Malachite Green. L-Aspartate-ß-hydroxamate, an asparagine analogue, was most effective in promoting the editing activity of AsnRS from Brugia malayi (BmAsnRS) and Staphylococcus epidermidis (SeAsnRS) with KM values close to 100 mM. The assay sensitivity was enhanced by the thiol agents, DTT and L-Cysteine, which significantly increased the turn-over of aminoacyl adenylate by BmAsnRS, but not SeAsnRS. The HTS assay was used to screen a library of 37,120 natural-product extracts for inhibitors of BmAsnRS. A small number of extracts that inhibited the pre-transfer editing by BmAsnRS was identified for future isolation of the active component(s). The principle of this assay can be applied to all enzymes having a pre- or post-editing activity.