The Site of Action of Oxazolidinone Antibiotics in Living Bacteria and in Human Mitochondria

Abstract

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.

The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning

“…In contrast, recent studies using an elegant in vivo cross-linking approach revealed the PTC as the bone fide drug target (12,13). These latter studies are consistent with the observation that drug resistance in both archaea and bacteria arises through mutation of nucleotides located within the PTC [ Fig.…”

“…also to the ribosomal elongation factor LepA (12,13). LepA is a translational GTPase that binds to POST state ribosomes inducing a back-translocation to the PRE state (37).…”

“…S4a). Generally, archaeal ribosomes are considered more ''eukaryotic-like'' with respect to their antibiotic specificities; however, this is not the case for the oxazolidinones as they have been shown to bind and/or inhibit both archaeal and bacterial (including mitochondrial) ribosomes but not to interact with human cytoplasmic ribosomes (13,14). In the H50S-linezolid structure (U.S. patent number 6,947,845 B2), the drug appears to bind deeper in the binding pocket when compared with the position in D50S, which could result from the shifted position of U2504.…”

Targeting Antibiotic Resistance

“…While oxazolidinones are protein synthesis inhibitors that bind to the ribosome, it has taken a number of years to identify the binding site and most likely mode of action 28. In 2008, two reported X‐ray co‐crystal structures of linezolid bound to 50S ribosomal subunits confirmed the previously established site of action and suggested a mode of action (Figure 4).…”

“…The first involves mutations in the 23S rRNA central loop of domain V, the peptidyl transferase center. While some of the mutated residues interact directly with the oxazolidinone, many do not but are instead used to stabilize the region surrounding the oxazolidinone 28. Mutations in these residues lead to small conformational changes of the linezolid binding pocket, which adversely affects drug binding.…”