Aminoglycosides, but not PTC124 (Ataluren), rescue nonsense mutations in the leptin receptor and in luciferase reporter genes

In rare cases, monogenetic obesity is caused by nonsense mutations in genes regulating energy balance. A key factor herein is the leptin receptor. Here, we focus on leptin receptor nonsense variants causing obesity, namely the human W31X, murine Y333X and rat Y763X mutations, and explored their susceptibilities to aminoglycoside and PTC124 mediated translational read-through in vitro. In a luciferase based assay, all mutations - when analysed within the mouse receptor - were prone to aminoglycoside mediated nonsense suppression with the highest susceptibility for W31X, followed by Y763X and Y333X. For the latter, the corresponding rodent models appear valuable for in vivo experiments. When W31X was studied in the human receptor, its superior read-through susceptibility – initially observed in the mouse receptor – was eliminated, likely due to the different nucleotide context surrounding the mutation in the two orthologues. The impact of the surrounding context on the read-through opens the possibility to discover novel sequence elements influencing nonsense suppression. As an alternative to toxic aminoglycosides, PTC124 was indicated as a superior nonsense suppressor but inconsistent data concerning its read-through activity are reported. PTC124 failed to rescue W31X as well as different nonsense mutated luciferase reporters, thus, challenging its ability to induce translational read-through.


The aminoglycoside antibiotic streptomycin lowers the efficacy of gentamicin to rescue mLEPR-b W31X
and mLEPR-b Y333X signalling. Standard cell culture medium is often supplemented with penicillin and streptomycin to prevent contaminations. Here, we performed the signalling assay with medium containing 100 U/ml penicillin and 100 µg/ml (170 µM) streptomycin (Biochrom) or medium only supplemented with 200 U/ml penicillin.
The presence streptomycin reduced the efficiency of gentamicin to rescue mLEPR-b W31X and mLEPRb Y333X signalling in HEK293 cells. When comparing the receptor activities measured in cells incubated with 6 nM leptin, only a doubling of mLEPR-b W31X signalling between 0 vs. 2.2 mM gentamicin was observed when medium contained streptomycin ( Supplementary Fig. 2 a). Without streptomycin, the gentamicin effect on mLEPR-b W31X signalling at 6 nM leptin was more than 8-fold ( Supplementary Fig.   2 a).
Streptomycin completely blocked the rescue effect of gentamicin on mLEPR-b Y333X ( Supplementary   Fig. 2 b). Only in the absence of streptomycin, gentamicin revived mLEPR-b Y333X activity ( Supplementary Fig. 2 b). This outcome can be explained by the fact that medium with streptomycin might promote 'basal' read-through at PTCs (Supplementary Fig. 2 c). Additionally, streptomycin could compete with gentamicin for the ribosomal binding site and thereby impairs the effect of gentamicin ( Supplementary Fig. 2 c). This outcome demonstrates the importance of optimal cell culture conditions to explore the activity of nonsense suppressor compounds.
The respective human ORFs -without the N-terminal His6-tag -were PCR amplified using the pDEST26 constructs as templates (primer pair 15 in Supplementary Table 1). The 3512 bp PCR products were cloned into pcDNA3.1 using the restriction enzymes NotI and HindIII. The functional efficiency of the pcDNA3.1 expression constructs was confirmed by the activation of hLEPR-b WT (inset Supplementary  Fig. 3). The expression of hLEPR-b W31X from pcDNA3.1 was not changing the susceptibility to nonsense suppression in comparison to the pDEST26 constructs (Fig. 4) since G418 and gentamicin did not rescue hLEPR-b W31X activity ( Supplementary Fig. 3).

Effect of G418 and PTC124 on secNLuc wild-type .
To explore whether G418 and PTC124 have off-target effects on secNLuc, we cloned secNLuc wild-type ORF into pcDNA5/FRT/TO. In the first place, we tested whether G418 and PTC124 disturb the bioluminescence reaction catalysed by secNLuc wild-type . To do so, medium from HEK293 cells transiently expressing secNLuc wild-type was collected and 500-fold diluted in water. Ten µl of this dilution were mixed with 10 µl NLuc assay buffer supplemented with raising amounts of G418 and PTC124 in 1 µl. Both compounds triggered a significant reduction of the bioluminescence signal indicating an inhibitory effect on secNLuc activity especially in the presence of PTC124 ( Supplementary Fig. 4 a and b). To further characterize the effect of PTC124 on the bioluminescence reaction, we added a constant concentration of 44 µM PTC124 to different amounts of secNLuc wild-type . Therefore, medium containing secNLuc protein was diluted (100-to 54,000-fold) and mixed with 44 µM PTC124. Independent from the secNLuc concentration, 44 µM PTC124 caused a consistent ~20% depression of the luminescence signal ( Supplementary Fig. 4 c and d).
In the next step, we tested whether G418 and PTC124 affect expression of secNLuc wild-type in transiently transfected HEK293 cells (e.g. via influencing transcription, transcript/protein stability or toxicity).
Medium was supplemented with rising concentrations of G418 of PTC124. After 24 h incubation, cells were washed with PBS and lysed with 20 µl NLuc assay buffer and 20 µl water. This lysate was 10-fold diluted in water and 1 µl of this mixture was added to 19 µl NLuc assay buffer to quantify luminescence.
We observed that PTC124 had no effect on secNLuc wild-type expression, whereas G418 induced a significant reduction most likely due to its toxic properties ( Supplementary Fig. 5 a and b).
One may speculate that the inhibitory activity of PTC124 on the bioluminescence reaction ( Supplementary Fig. 4) could mask a translational read-through effect. The consistent ~20% depression of the bioluminescence signal over a large secNLuc wild-type concentration range illustrates that only a