Potency estimates depend on the kinetic endpoint and assay configuration. The authors calculated a K50% of 1.3 μM for doubling the single-round elongation-cycle time, versus about 3 μM for halving EF-G cycling in a factor-limited multiple-turnover assay. These are biochemical kinetic estimates, not whole-cell IC50 values. [1]
Single-round peptide elongation and translocation kinetics
Biochemical assay · Purified E. coli MRE600 ribosomes programmed for fMet-Leu-Phe synthesis; excess EF-G at 37 °C in polymix buffer. [1]
FA produced a slow component in tripeptide formation relative to the no-FA comparator. Results at 5 and 20 μM EF-G were similar. Dipeptide and pyrene-mRNA measurements constrained the inference: initial mRNA movement remained rapid, whereas later steps and EF-G release were delayed. The reported K50% is a kinetic-model estimate for doubling average elongation-cycle time.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0–2 mM FA after equal-volume mixing; final EF-G 5 or 20 μM in tripeptide experiments. Pyrene-mRNA control used 0–400 μM final FA.
- Exposure duration
- Time-resolved reactions, from milliseconds into seconds; no single fixed treatment duration.
- Method
- Quench-flow with HPLC/radiometric peptide quantification; stopped-flow pyrene-mRNA fluorescence as an orthogonal movement control.
- Reported limits
- Drug-stock solvent and replicate n for the selected measurements were not specified in the read assay sections. Translation factors are assay components, not combination drugs.
Methods and source locations
Compound reported in the paper: Fusidic acid sodium salt; Compound label in the paper: FA
Evidence for the reported sodium-salt reagent in a purified bacterial translation system; no whole-cell potency, therapeutic dose or Adooq lot validation is implied.
Materials and Methods: Reagents and Buffer Conditions; single-round translocation and fluorescence-labelled mRNA subsections; Results: corresponding subsections; Figs. 3, 5, 6; Discussion: How Sensitive Is the Translocating Ribosome to Fusidic Acid?
Concentration calculated to double the average elongation-cycle time in the single-round tripeptide system: Authors’ calculation using Equation 44 and fitted parameters; not a cellular IC50 or a directly titrated binding constant. — Discussion: How Sensitive Is the Translocating Ribosome to Fusidic Acid?; Equation 44; Fig. 5; Table 1
Timing of the initial fluorescence rise and subsequent fall associated with mRNA movement: Stopped-flow fluorescence; the final fluorescence amplitude changed with FA, so amplitude change was not interpreted as slower mRNA movement. — Results: FA Inhibition of Translocation by EF-G in Single Round Mode with Fluorescence-labeled mRNA; Fig. 6A–B
Multiple-turnover EF-G cycling assay
Biochemical assay · Purified E. coli translation system with ribosomes in excess over EF-G: final ribosomes 2 μM and EF-G 0.05 μM, 37 °C. [1]
At 20 μM FA, EF-G cycling slowed relative to the no-FA control after an initial transient. From the steady-state slope ratio, the authors estimated K50% at about 3 μM. They attributed its difference from the single-round estimate to slow EF-G binding under factor-limited cycling conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0 or 20 μM FA after mixing.
- Exposure duration
- Curve fits used up to 3 s without FA and 8 s with FA.
- Method
- Equal-volume rapid mixing; HPLC and online radiometry followed fMet-Leu-Phe formation per active EF-G molecule.
- Reported limits
- Drug-stock solvent and replicate n for the selected measurements were not specified in the read assay sections. Translation factors are assay components, not combination drugs.
Methods and source locations
Compound reported in the paper: Fusidic acid sodium salt; Compound label in the paper: FA
Evidence for the reported sodium-salt reagent in a purified bacterial translation system; no whole-cell potency, therapeutic dose or Adooq lot validation is implied.
Materials and Methods and Results: FA Inhibition of Translocation by EF-G in Multicycle Mode; Fig. 7
Concentration estimated to halve the EF-G cycling rate: Estimated from the ratio of linear portions of the no-FA and 20 μM FA curves using a simple uncompetitive model; not a dose-response cellular IC50. — Results: FA Inhibition of Translocation by EF-G in Multicycle Mode; Fig. 7
Paired ribosome-splitting and GTP-consumption measurements
Biochemical assay · Purified E. coli MRE600 post-termination ribosomes; final ribosomes 1 μM, EF-G 0.5 μM, RRF 30 μM and [3H]-GTP 20 μM, at 37 °C. [2]
FA delayed ribosome splitting and GTP hydrolysis, but the GTP cost per successful splitting event remained close to 3.5 across the tested range. The ratio was corrected for subsequent GTP hydrolysis on released 50S subunits by extrapolation to zero time. These results support retention of a splitting-competent complex during FA stalling.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0–150 μM FA; no-FA control included.
- Exposure duration
- Time-course sampling; Figure 3 displays reactions through approximately 400 s.
- Method
- Matched stopped-flow light-scattering measurements of splitting and quench-flow/HPLC measurements of GTP hydrolysis under the same conditions.
- Reported limits
- Drug-stock solvent and replicate n for the selected measurements were not specified in the read assay sections. Translation factors are assay components, not combination drugs.
Methods and source locations
Compound reported in the paper: Fusidic acid sodium salt; Compound label in the paper: FA
Evidence for the reported sodium-salt reagent in a purified bacterial translation system; no whole-cell potency, therapeutic dose or Adooq lot validation is implied.
Materials and Methods: GTP hydrolysis and ribosome splitting measurements; Results: The average number of GTP molecules per ribosome splitting does not vary with the FA concentration; Fig. 3A–D; Discussion
GTP molecules hydrolysed per ribosome splitting event, corrected for the 50S side reaction: Ratio of GTP hydrolysed to ribosomes split plotted versus time; intercepts of linear fits estimate the zero-time stoichiometry. Figure points vary; the authors describe the overall consumption as essentially invariant. — Results: The average number of GTP molecules per ribosome splitting does not vary with the FA concentration; Fig. 3A–D
