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 reporter gene assays ͉ high-throughput screening ͉ Ataluren

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

“…S1C) (21). It has been suggested that ataluren is an inhibitor of firefly luciferase and that its reported nonsense suppression activity simply reflects stabilization of the luciferase enzyme (18,22). We consider this claim to be spurious because (i) ataluren-mediated readthrough has been observed in numerous other systems ( Fig.…”

“…1). This result was attributable to readthrough of the reporter mRNA's premature UGA codon, and not protein stabilization (18), because ataluren treatment of 293H cells stably expressing WT NanoLuc had no effect on NanoLuc activity (Fig. 1B).…”

“…In the latter, Translarna clinical trials have demonstrated restoration of full-length functional protein in Duchenne muscular dystrophy and cystic fibrosis nonsense mutation patients (10,11,13,15,16). However, a lack of activity has been reported for some cell systems (17,18).…”

Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4

“…From the primary screen, compounds that gave rise to a signal Ͼ2.2-fold (Ϸ5,000 compounds) over the plate averageassuming that most compounds are inactive and can serve as controls (31)-were selected for reanalysis in triplicate. Measurements with these putative hit compounds were collected and averaged, and those that activated the luciferase signal 2.5-fold over DMSO-treated controls (Ϸ1,000 compounds) were counterscreened in a cell-based SV40-driven firefly luciferase assay to rule out false-positives that directly and nonspecifically activate luciferase reporters (32). Compounds that reproducibly hit in the Nanog assay but did not hit in the SV40 assay were further analyzed in a dose-response format to identify the optimal concentration for activity ( Fig.…”

Nonsense suppression activity of PTC124 (ataluren)

“…are employees or consultants of PTC Therapeutics, Inc. 1 To whom correspondence should be addressed. E-mail: speltz@ptcbio.com.…”