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. Author manuscript; available in PMC: 2021 Jul 14.
Published in final edited form as: SLAS Discov. 2020 Aug 4;26(1):1–16. doi: 10.1177/2472555220945284

Figure 2: Development of GPCR-specific secondary assays to enable immediate scaffold validation and compound characterization:

Figure 2:

(A) Design and mechanism of TR-FRET based binding assay employing SNAP-tag technology and fluorescein-labeled secretin peptide (Fluo-Sec); (B) Saturation binding curve of Fluo-Sec determining dissociation constant Kd; (C) Competition binding curve of Sec-FL; LanthaScreen ratios normalized to Fluo-Sec bound. (D) Design and mechanism of β-arrestin-2 recruitment assay exploiting NanoBiT (Promega) technology; (E) Real-time luminescence after ligand addition to evaluate best time point for endpoint read; (F) Dose-response curve of Sec-FL at 3 min (transiently expressing HEK-293 cells); (G) Normalized dose-response curve of Sec-FL (HEK-293 SCTR-SmBiT LgBiT-ARRB2 cell clone, n = 4), raw data signal-to-background ratio (S/B) over 17. Relative luminescence units (RLU) normalized to Sec-FL; graphs plotted using GraphPad Prism; data points shown as mean ± SEM.