Fig. 7.

Validation of the dual-readout F² assay in an uHTS format. The Mcl-1 F² assay was validated for uHTS using a 102,255 compounds library from Molecular Library Screening Center Network as an example. Library compounds were added to the 1,536-well plates containing reaction buffer with TMR-Noxa peptide and Mcl-1 and incubated at room temperature for 2 h. The TR-FRET signal and the FP signal from each well containing a library compound were measured and plotted. (A) The percentage of inhibition from TR-FRET readout was plotted against the FI of the compound. FI of the compound was expressed as FOC and calculated by the fold increase of FI at 545 nm from compound wells over that from vehicle control wells for each plate. (B) The percentage of inhibition from FP readout was calculated and plotted against the FI of the compounds expressed as FOC. FOC was calculated by the fold increase of the p-channel FI counts from a compound well over that from vehicle control wells for each plate. Compounds that showed percentage of inhibition > 50 in both TR-FRET and FP readout were considered as potential hits. Compounds with FI ≤ 0.2, or FI ≥ 2 were defined as FI artifacts. (C) FI of the library compounds from FP readouts was plotted against that from TR-FRET readouts. (D) The percentage of inhibition from FP readout was plotted against that from TR-FRET readout. FOC, fold of control.