Figure 3. Reconfirmation and secondary assays of γ-globin inducer compounds.

Panel A) Reconfirmation of γ-luc inducibility by 232 actives from primary HTS. Four secondary assays were employed including two reconfirmation assays for firefly luciferase induction and cytotoxicity, and two specificity assays for Renilla luciferase activity and luciferase enzyme modulators. All assays were a 10-point dose-response. A summary of firefly induction is shown in this figure. 211 of the 232 compounds had firefly induction of 2-fold or higher; a 90% reconfirmation rate. Panels B-D) Performance of seven γ-firefly inducers in the initial four secondary assays – detailed 10-point dose-response data. Comparison of compound activity from dose-response data is shown for firefly and Renilla luciferase activity (Panel B), purified firefly luciferase enzyme inhibition (Panel C), and cytotoxicity (Panel D). Assays were performed as described in Materials and Methods.