Fig. 6: DISCo-BRET demonstrates significantly slower on-rate kinetics at FFA1 site one when a compound is bound to site two.

Sequential binding experiments were conducted by treating first with red TUG-2591 (100 nM), then with green TUG-2597 (316 nM) (A), and data were plotted as either the 690 nm / 540 nm (B) or the 540 nm / 450 nm (C) luminescent emission ratios. Experiments were carried out either as control experiments or in the presence of TUG-905 (10 μΜ) to prevent binding of TUG-2591. Specific BRET was obtained by subtracting the non-specific BRET ratio obtained in cells that had been pre-treated with both TUG-905 (10 μΜ) and T360 (10 μΜ). Data in (B, C) are mean ± SEM from N = 4 (Control) or N = 3 (TUG-905) independent experiments. Comparable sequential binding experiments were conducted treating first with TUG-2597 (316 nM), then with TUG-2591 (100 nM) (D−F). Data in (E, F) are mean ± SEM from N = 5 (Control) or N = 3 (TUG-905) independent experiments. G Sequential binding experiments adding first TUG-2597 (316 nM) followed by increasing concentrations of TUG-2591. Data are mean ± SEM from N = 3 independent experiments. H The TUG-2591 association portion of these experiments are isolated and fit to a multiple concentration of labelled ligand binding association model. Data are mean ± SEM from N = 3 independent experiments. I Comparable NanoBRET association experiments using an N terminal Nluc tagged FFA1 construct and multiple concentrations of TUG-2591 are fit to a multiple concentration of labelled ligand binding association model. Data are mean ± SEM from N = 4 independent experiments. Source data are provided as a Source Data file.