Reply to Peltz et al: Post-translational stabilization of the firefly luciferase reporter by PTC124 (Ataluren)

The IC₅₀ of an inhibitor varies depending on assay conditions such as substrate concentration (1), as depicted for PTC124 in Fig. 1A of Peltz et al.'s letter (2). Steady-Glo and Bright-Glo differ in luciferase substrate composition, making comparison of inhibitor potency using these commercial formulations meaningless and inconsequential to inhibitor-based stabilization of the reporter occurring intracellularly (Fig. 1 in ref. 3). Accurate determination of the affinity of the enzyme-inhibitor complex is obtained through the constant K_I (1, 4), which we estimated to be ≈10 nM for PTC124 against FLuc (using [S]∼K_M) (3). Further, using a classic pharmacological approach (5)— strong correlation between inhibition of isolated FLuc and potency in cell-based assays of PTC124 and analogs (Fig. 4C in ref. 3)—we unmistakably demonstrate that increases in cell-based FLuc activity is due to PTC124 interaction with the FLuc protein.

Peltz et al.'s inability to reproduce our findings in the cell-based FLuc assay (Fig. 1B in ref. 2) is likely due to their omission of a critical step in our stated protocol, which removes PTC124, a potent inhibitor, by wash steps prior to detection (3). This allows measurement of the true FLuc activity without the complications illustrated by Peltz et al. (Fig. 1 in ref. 2) and supports our finding that the observed response is highly biased by FLuc inhibitory activity. Furthermore, our RLuc-based readthrough assay, which showed that PTC124 was inactive, demonstrated sufficient sensitivity to gentamicin (2-fold activation), an RLuc inhibitor (compd.4; ≈2.5-fold), and a transcriptional activator (≈7-fold; Fig. 3, in ref. 3)—sensitivity not demonstrated in the assay conducted by Welch et al. (6).

Thus by all standard enzymological and pharmacological measures, PTC124 potently binds to FLuc leading to increased cell-based activity via post-translational reporter stabilization.