The minor gentamicin complex component, X2, is a potent premature stop codon readthrough molecule with therapeutic potential

Figures

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  Fig 1. Gentamicin complex and individual components cause readthrough of a native p53-NS allele in HDQ-P1 cells. p53 was detected using a previously described MSD immunoassay (A and B) or by western blot hybridization analysis (C). (A) Readthrough activity of gentamicin complex components is similar to that of the gentamicin complex. (B) Gentamicin X2 and G418 are potent readthrough compounds as compared to the gentamicin complex. Data in Fig 1A and 1B are from a representative experiment. Summarized data from all readthrough experiments can be found in Table 1.

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  Fig 2. Statistical comparison of selected aminoglycosides. Graphs show average values for at least three independent experiments (open circles) for each aminoglycoside indicated on the y-axis. Error bars indicate the 95% confidence limits for each average. The title of each graph indicates the average measurement or the average calculated ratio. A-C are potency averages from the three readthrough assays; D-F are averages for the three cytotoxicity assays and G-O show the mean ratios between the readthrough and the cytotoxicity assays. All values used for calculating averages can be found in S1 File.

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  Fig 3. G418 and gentamicin X2 have similar pharmacokinetics in the rat. (A) G418 and gentamicin X2 were administered by subcutaneous injection to rats at a dose of 10 mg/kg. Plasma was collected at the indicated time points and drug levels were determined. Symbols represent the mean of three rats ± standard deviation (SD) per group. (B) G418 and gentamicin X2 accumulate in kidney tissues. The concentration of aminoglycoside in the tissue (μg compound per gram wet weight of tissue) at 24 hours post-dose is the mean ± SD of three rats. Levels of aminoglycosides were at or below the lower limit of quantification. * p < 0.05 (Student’s t-test).

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  Fig 4. Gentamicin X2 was better tolerated than G418. When administered at a dose of 20 mg/kg G418 (A) caused a greater decrease in body weight than did gentamicin X2 (B) when administered at a dose of 40 mg/kg. Rat body weights can be found in S1 Table.

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  Fig 5. Elevated markers of kidney damage with G418 but not with gentamicin X2. After 9 days of dosing, urine was collected from vehicle treated and the highest dose groups of G418 and gentamicin X2 and analyzed for the indicated markers of kidney damage. Albumin, clusterin cyctatin and osteopontin were increased in the urine of rats dosed with G418 (20mg/kg) but not increased in urine from rats dosed with gentamicin X2 (40 mg/kg). Alpha-GST levelsshowed the greatest decrease in rats dosed with 20 mg/kg G418 compared to 40 mg/kg gentamicin X2. Lipocalin was not affected by either drug.

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  Fig 6. Structure of G418 and gentamicin complex components. The structure on the top left and the table show the structural differences between gentamicin complex components and G418. The structure of the other two gentamicin complex components are shown in the lower two drawings.