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Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression
Douglas S Auld1, Natasha Thorne, William F Maguire
1NIH Chemical Genomics Center, National Institutes of Health, Bethesda, MD 20892-3370, USA.
Abstract:
High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87-91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC(50) = 7 +/- 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.
Insights
Compounds can interfere with reporter enzymes like firefly luciferase (FLuc) in drug discovery assays. This study shows PTC124 directly inhibits FLuc, not suppressing nonsense codons as previously thought.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- High-throughput screening (HTS) assays often use reporter enzymes like firefly luciferase (FLuc) to measure target activity.
- Compounds can directly modulate reporter enzyme activity, leading to false positives or negatives in HTS.
- Previous studies identified 3,5-diaryl-oxadiazole compounds as potential FLuc inhibitors.
Purpose of the Study:
- To evaluate 3,5-diaryl-oxadiazole compounds, including PTC124, for direct activity against firefly luciferase (FLuc).
- To investigate whether the observed nonsense codon suppression activity of PTC124 is a direct effect on FLuc.
- To emphasize the importance of control assays in HTS to avoid misinterpretation of results.
Main Methods:
- Tested a series of 3,5-diaryl-oxadiazole compounds for FLuc inhibition.
- Assessed the activity of these compounds in a FLuc reporter cell-based nonsense codon assay.
- Evaluated the compounds' activity against Renilla reniformis luciferase (RLuc) and in an RLuc reporter assay.
Main Results:
- The inhibitory potency of tested compounds against FLuc directly correlated with their activity in the FLuc reporter assay.
- PTC124 was identified as a potent FLuc inhibitor with an IC(50) of 7 +/- 1 nM.
- PTC124 and related compounds did not exhibit nonsense codon suppression activity with RLuc, indicating FLuc is a direct target.
Conclusions:
- The initial discovery of PTC124's nonsense codon suppression activity was likely due to direct inhibition of the firefly luciferase reporter.
- Firefly luciferase appears to be a direct molecular target of PTC124.
- Understanding compound-reporter interactions and using appropriate controls are crucial for accurate HTS data interpretation.
