A lack of premature termination codon read-through efficacy of PTC124 (Ataluren) in a diverse array of reporter assays

Abstract

The drug molecule PTC124 (Ataluren) has been described as a read-through agent, capable of suppressing premature termination codons (PTCs) and restoring functional protein production from genes disrupted by nonsense mutations. Following the discovery of PTC124 there was some controversy regarding its mechanism of action with two reports attributing its activity to an off-target effect on the Firefly luciferase (FLuc) reporter used in the development of the molecule. Despite questions remaining as to its mechanism of action, development of PTC124 continued into the clinic and it is being actively pursued as a potential nonsense mutation therapy. To thoroughly test the ability of PTC124 to read through nonsense mutations, we conducted a detailed assessment comparing the efficacy of PTC124 with the classical aminoglycoside antibiotic read-through agent geneticin (G418) across a diverse range of in vitro reporter assays. We can confirm the off-target FLuc activity of PTC124 but found that, while G418 exhibits varying activity in every read-through assay, there is no evidence of activity for PTC124.

Figure 1. Differing activity of PTC124 and G418 in FLuc reporter cell lines.

(A) Stop codon-containing FLuc activity in ADXC8 cells treated for 24 hours with G418 (closed circles) or PTC124 (open circles) which were not washed of compound prior to addition of luciferase detection buffer. (B) FLuc activity in ADXC8 cells in which compound was washed off prior to addition of detection buffer. Both compounds were also tested under the same conditions in WT-FLuc cells using the same (C) no wash and, (D) wash protocol. All data points represent mean ± standard deviation (n = 5).

Figure 2. Lack of PTC124 efficacy with a stably expressed β-Galactosidase PTC-reporter.

(A) Incubation with G418 for 24 hours leads to a dose-dependent increase in enzyme activity of stably transfected PTC-β-Gal. (B) 24-hour incubation with PTC124 at concentrations in excess of its reported effective concentration range of 0.1–30 µM has no effect. All data points represent mean ± standard deviation (n = 5).

Figure 3. Lack of PTC124 efficacy with a transiently transfected RLuc PTC-reporter.

(A) Incubation with G418 for 24 hours leads to a dose-dependent increase in enzyme activity of transiently transfected PTC-RLuc. (B) 24-hour incubation with PTC124 at concentrations in excess of its reported effective concentration range of 0.1–0 µM has no effect. All data points represent mean ± standard deviation (n = 5).

Figure 4. Lack of PTC124 efficacy with transiently transfected collagen VII PTC reporters.

Dose-dependent increases in detectable protein with G418 but not PTC124 in R578X (A and B) and Q251X (C and D) PTC collagen VII constructs. All data points represent mean ± standard deviation (n = 4).

Figure 5. PTC124 does not increase detectable K6a-YFP production for any PTC sequence.

Twelve PTC mutant K6a-YFP constructs were generated covering all potential PTC sequence contexts including variations in the +1 base. All constructs were transiently transfected into AD293 cells, which were subsequently treated with DMSO control, 200 µM G418, or 3 µM PTC124 for 24 hours before Western blots were conducted to determine presence of YFP, GFP transfection control, and K18 loading controls.