Evernimicin binds exclusively to the 50S ribosomal subunit and inhibits translation in cell-free systems derived from both gram-positive and gram-negative bacteria - PubMed (original) (raw)

Evernimicin binds exclusively to the 50S ribosomal subunit and inhibits translation in cell-free systems derived from both gram-positive and gram-negative bacteria

P M McNicholas et al. Antimicrob Agents Chemother. 2000 May.

Abstract

Evernimicin (SCH 27899) is a new antibiotic with activity against a wide spectrum of gram-positive bacteria and activity against some gram-negative bacteria. Previous metabolic labeling studies indicated that evernimicin specifically inhibited protein synthesis in Staphylococcus aureus. Using a susceptible Escherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli. In cell-free translation assays with extracts from either E. coli or S. aureus, evernimicin had a 50% inhibitory concentration of approximately 125 nM. In contrast, cell-free systems derived from wheat germ and rabbit reticulocytes were inhibited only by very high levels of evernimicin. Evernimicin did not promote transcript misreading. [(14)C]evernimicin specifically bound to the 50S subunit from E. coli. Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli and S. aureus and 50S subunits from E. coli returned dissociation constants of 84, 86, and 160 nM, respectively. In binding experiments, performed in the presence of excess quantities of a selection of antibiotics known to bind to the 50S subunit, only the structurally similar drug avilamycin blocked binding of [(14)C]evernimicin to ribosomes.

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Figures

FIG. 1

FIG. 1

Structures of avilamycin and evernimicin.

FIG. 2

FIG. 2

Effect of evernimicin on incorporation of [14C]isoleucine (A) [14C]uracil (B), and [14C]thymidine (C) by E. coli HS227-5. Evernimicin was added to exponential-phase cultures at the time indicated by the arrow at the following multiples of the MIC (4 μg/ml): 0.5× (solid triangles), 1× (solid squares), and 2× (solid diamonds). Results for reactions with chloramphenicol at 20 μg/ml (open triangles) and no antibiotic (controls; open squares) were also included. The data were normalized, to correct for differences in growth rate resulting from addition of test antibiotics, by dividing the counts obtained at a given time point by the OD600.

FIG. 3

FIG. 3

Effect of evernimicin on in vitro translation reactions with various mRNA templates, ribosomes washed with salt at low concentrations, and S100 extracts from E. coli A19 and S. aureus RN450. Incorporation of the various 14C-labeled amino acids into TCA-precipitable material is expressed as a percentage of that in the control (no antibiotic) reaction. (A) Poly(U)-based reaction with components from E. coli (solid diamonds) and S. aureus (open squares) and a poly(A)-based reaction with components from E. coli (solid triangles) and S. aureus (open circles). (B) E. coli components with poly(U) (solid diamonds) and phage MS2 (solid squares) templates.

FIG. 4

FIG. 4

Nonlinear regression analysis of [14C]evernimicin binding to 70S ribosomes from E. coli A19 (open squares) and S. aureus RN450 (open circles) washed with salt at low concentrations and purified 50S subunits from E. coli A19 (crosses).

FIG. 5

FIG. 5

Binding of [14C]evernimicin to ribosomal subunits from E. coli A19. 70S ribosomes (115 pmol) washed with salt at low concentrations and [14C]evernimicin (98 pmol) were incubated in buffer with a low concentration of magnesium, and the resultant antibiotic-subunit complexes were resolved on a 10 to 30% sucrose gradient. The gradient was fractionated from the bottom and radioactivity (solid line) and the A260 (dashed line) were determined for each fraction. The peaks corresponding to the 30S and 50S subunits are labeled.

FIG. 6

FIG. 6

Competition between avilamycin and evernimicin for binding to 70S ribosomes from E. coli A19 washed with salt at low concentrations. Various amounts of unlabeled avilamycin (squares) and evernimicin (diamonds) were mixed with a fixed amount (0.156 μg, 98 pmol) of [14C]evernimicin and incubated with 70S ribosomes from E. coli washed with salt at low concentrations. Following binding the amount of bound antibiotic was determined.

References

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