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. 2022 Aug 9;23(9):3822–3830. doi: 10.1021/acs.biomac.2c00671

Figure 2.

Figure 2

Lifetime FRET HTS. (A) Pilot screening with LOPAC library. Compounds that reduced the FRET efficiency below 3SD (black line) were selected for further characterization. Nine hit compounds with reproducible FRET change were identified from the pilot screens (red). (B) Dose response curves of three HbS hit compounds (panel A, red circles). Fluorescently labeled HbS samples were dispensed into a 1536-well plate and treated separately with DMSO or the three hit compounds, chlormezanone, gabazine, and phosphoramidon disodium, in a dose-dependent manner from 0.1 to 1000 μM for 2 h at room temperature and then vacuum degassed for 10 min, and then donor lifetimes were measured using a fluorescence plate reader. (C) Effective diameter of HbS in the presence and absence of hit compounds from DLS measured at 90°. HbS samples (25 μM) were incubated separately with hit compounds (100 μM) or DMSO only for 2 h at room temperature and degassed with argon for 5 min, and then DLS measurements were taken at room temperature. Hypothesis testing was performed using parametric ANOVA with Dunnett’s multiple comparison testing. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.