title: Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.
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.
authors.affiliation: NIH Chemical Genomics Center, National Institutes of Health, Bethesda, MD 20892-3370, USA.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Auld
authors.forename: Natasha
authors.initials: N
authors.lastname: Thorne
authors.forename: William F
authors.initials: WF
authors.lastname: Maguire
authors.forename: James
authors.initials: J
authors.lastname: Inglese
mesh_terms: D000818:Animals; D018389:Codon, Nonsense / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D049409:Luciferases, Firefly / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D010069:Oxadiazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D018389:Codon, Nonsense; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D010069:Oxadiazoles; D049409:Luciferases, Firefly; D006655:Histone Deacetylases; C515878:ataluren