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.

Fig. 1.

Posttranslational reporter stabilization in gene activation assays. (A) Luciferase reporter protein half-life differs in cells not treated (Upper) and treated (Lower) with an inhibitor of the luciferase enzyme. (Lower) Interaction of the inhibitor with the luciferase protein stabilizes the protein, thereby increasing the protein half-life and allowing increased accumulation of reporter protein in the cell. (B) Level of apparent reporter activation depends on properties of the luciferase inhibitor and the assay detection protocol used. After incubation with compound, the cells are lysed, and luciferase detection reagents are added, which may result in displacement of the inhibitor from the reporter (i, illustrated in the schematic of cells) or the inhibitor may remain bound to the reporter (ii, illustrated in the schematic of cells). Shown is the theoretically observed activity of the reporter in the cell-based assay (blue lines) and activity against the reporter enzyme determined in a biochemical assay where substrates are at low concentrations (e.g., ≤K_m; orange lines). (Insets) Increase in enzyme concentration in the cell-based assay as a result of inhibitor-based stabilization. (i) If the cells are treated with a compound that acts as a reversible competitive inhibitor of luciferase (orange curve), after interaction and stabilization of the luciferase protein (Inset), the compound can be effectively competed off the luciferase enzyme after cell lysis and treatment with detection reagent containing excess substrates. In this scenario, apparent activation is observed in the cell-based assay (blue curve) that parallels the inhibition of the reporter enzyme in a cell-free, biochemical assay (orange curve). Alternatively, reversible noncompetitive inhibitors may be removed by washing the cells free of compound before detection resulting in the same apparent activation. (ii) If the cells are treated and detected in the presence of a compound that acts as an irreversible or a noncompetitive inhibitor of the luciferase enzyme (orange curve) where the inhibitor is not removed before detection, stabilization of the reporter enzyme still occurs (Inset), but in this scenario inhibition is likely observed (blue curve) because the inhibitor is not displaced from the enzyme during detection. More complex apparent activation behavior such as bell-shaped concentration–response curves may also occur, which are further detailed in SI Appendix.

Fig. 2.

Structures of compounds used to evaluate the assays. Shown are the 3,5-diaryl-oxadiazole core (1) present in PTC124 (1a) and analogs synthesized in this work (1 b–k), aminoglycosides G418 (2a) and gentamicin (2b), the HDAC inhibitor MS-275 (3), the RLuc inhibitor 4-methyl-N-(phenylmethyl)benzenesulfonamide (4), and the benzthiazole-based FLuc inhibitors (5 a and b).

Fig. 3.

PTC124 activity depends on the reporter. (A) (i) (Upper) FLuc activity from Grip-Tite 293 cells transfected with the pFLuc190^UGA construct and treated with compound 1a (PTC124) for 24 h (PTC124; open circles) shows concentration-dependent activation. (Lower) Activity of purified FLuc enzyme (with K_m concentrations of substrates) shows concentration-dependent inhibition with compound 1a treatment (filled circles). (ii) Aminoglycoside treatment for 24 h with 600 μg/mL G418 or 1 mg/mL gentamicin alone or in combination. Percentage activity of cells with vehicle alone is significantly different from that of cells treated with G418, gentamicin, or both (unpaired t test; *, P < 0.0001 for each comparison; data from 168 assay wells). (iii) FLuc activity from cells transfected with the pFLuc190^UGA construct and treated with compound 3, MS-275, for 24 h. (B) (i) RLuc activity is not modulated by compound 1a (PTC124), either in the cell-based assay using Grip-Tite 293 cells transfected with the pRLuc110^UGA construct and treated for 72 h with compound (Upper, open circles) or in the biochemical assay using purified RLuc enzyme (with K_m concentrations of coelenterazine substrate; Lower, filled circles). Compound 4 shows activation of RLuc in the cell-based assay (Upper, open squares) after 72-h treatment with compound, and inhibition of purified RLuc enzyme (Lower, filled squares). (ii) Antibiotic treatment for 72 h with G418 or gentamicin alone or in combination. Similar to what is seen for pFLuc190^UGA experiments, percentage activity for cells without antibiotic treatment is significantly different from that of cells treated with G418, gentamicin, or both (unpaired t test; *, P < 0.0001 for each comparison; data from 168 assay wells). (iii) RLuc activity from cells transfected with the pRLuc110^UGA construct and treated with compound 3 for 72 h. Data from duplicate or quadruplicate determinations from experiments performed on separate days (n = 2 or 3) are expressed as the percentage activity ± SEM.

Fig. 4.

Relationship of PTC124 cell-based activity with FLuc inhibition potency. (A) (Upper) FLuc activity from Grip-Tite 293 cells transfected with the pFLuc190^UGA construct and treated with PTC124 (1a, open circles) and PTC124-analogs 1h (open squares) and 1k (open triangles). (Lower) Activity of purified FLuc enzyme with K_m concentrations of substrates with treatment of compound 1a (filled circles), 1h (filled squares), and 1k (filled triangles). (B) Activity of benzthiazole-based FLuc inhibitors (5a, circles; 5b, squares) in the cell-based assay with 293 cells transfected with the pFLuc190^UGA construct (Upper) and in the purified FLuc assay (Lower). (C) Pharmacological correlation of the EC₅₀ for activation in the pFLuc190^UGA cell-based assay with the IC₅₀ derived against purified luciferase enzyme (r² = 0.85; dashed lines represent the 95% confidence intervals). Mean ± SEM are from two to four replicates that were repeated on 2 separate days.

Fig. 5.

PTC124 selectively decreases proteolysis of FLuc by trypsin. Purified luciferase enzymes were incubated with trypsin in the presence (filled symbols) and absence (open symbols) of 2 μM PTC124. Aliquots were removed and diluted (90-fold) with the appropriate luciferase detection buffer. Data shown are the mean ± SE from three determinations. Circles, Fluc; triangles, RLuc. Values for t_1/2 are: RLuc, 4 ± 0.1 min; RLuc with PTC124, 5 ± 0.2 min; Luc, 8 ± 0.4 min; Luc with PTC124, 17 ± 0.9 min.