ABSTRACT
Novel antimicrobials that effectively inhibit bacterial growth are essential to fight the growing threat of antibiotic resistance. A promising target is the bacterial ribosome, a 2.5 MDa organelle susceptible to several biorthogonal modes of action used by different classes of antibiotics. To promote the discovery of unique inhibitors, we have miniaturized a coupled transcription/translation assay using E. coli and applied it to screen a natural product library of ~30 000 extracts. We significantly reduced the scale of the assay to 2 µL in a 1536-well plate format and decreased the effective concentration of costly reagents. The improved assay returned 1327 hits (4.6% hit rate) with %CV and Z' values of 8.5% and 0.74, respectively. This assay represents a significant advance in molecular screening, both in miniaturization and its application to a natural product extract library, and we intend to apply it to a broad array of pathogenic microbes in the search for novel anti-infective agents.
Subject(s)
Biological Products/pharmacology , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Anti-Infective Agents/pharmacology , Escherichia coli/drug effects , Escherichia coli/genetics , Luciferases/genetics , Miniaturization/methods , Protein Biosynthesis/drug effects , Ribosomes/drug effects , Ribosomes/genetics , Small Molecule Libraries , Transcription, Genetic/drug effectsABSTRACT
The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.
Subject(s)
Mitochondria/drug effects , Oxazolidinones/pharmacology , Peptidyl Transferases/drug effects , Protein Synthesis Inhibitors/pharmacology , RNA, Ribosomal/drug effects , Software , Acetamides , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Binding Sites/drug effects , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/pharmacology , Cytoplasm/drug effects , Cytoplasm/enzymology , Drug Resistance/genetics , Escherichia coli/drug effects , Escherichia coli/enzymology , Humans , Linezolid , Mitochondria/enzymology , Models, Molecular , Molecular Structure , Mutation/genetics , Oxazolidinones/chemistry , Peptidyl Transferases/metabolism , Protein Synthesis Inhibitors/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Ribosomal/metabolism , RNA, Ribosomal, 23S , RNA, Transfer, Amino Acyl/antagonists & inhibitors , RNA, Transfer, Amino Acyl/metabolism , Staining and Labeling , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymologyABSTRACT
The oxazolidinones are a novel class of antibiotics that inhibit initiation of protein synthesis in bacteria. In order to investigate their novel mechanism of action, the interactions of several oxazolidinones with bacterial 70S ribosomes, 50S subunits, and 30S subunits have been characterized by (1)H nuclear magnetic resonance (NMR) line-broadening analyses and transferred nuclear Overhauser enhancement (TRNOE) experiments. PNU-177553 and PNU-100592 (eperezolid) and their corresponding enantiomers, PNU-184414 and PNU-107112, were studied. The dissociation constants were determined to be 94 +/- 44 microM and 195 +/- 40 microM for PNU-177553 and eperezolid, respectively. There was a approximately 4-fold decrease in affinity for their corresponding enantiomers. The NMR-derived dissociation constants are consistent with their antibacterial activity. PNU-177553 and eperezolid were found to bind only to the 50S subunit, with similar affinity as to the 70S ribosome, and to have no affinity for the 30S subunit. Specific binding of PNU-177553 was further confirmed in TRNOE experiments in which positive NOEs observed for the small molecule alone were changed to negative NOEs in the presence of bacterial 70S ribosomes. The observed NOEs indicated that PNU-177553 did not adopt a significantly different conformation when bound to the 70S ribosome, compared to the extended conformation that exists when free in solution. Since this is likeliest the case for each of the four compounds included in this study, the A ring C5 side chain may be positioned in the proper orientation for antibacterial activity in PNU-177553 and eperezolid but not in their inactive enantiomers.
