Fig. 2: Identification of TUG-2591 as a red fluorescent tracer binding to FFA1 site one.
A Chemical structures of putative red fluorescent tracers for site one of FFA1. Compounds are based on a previously described green tracer, TUG-1460, with the linkers used as well as the position and identity of the red fluorophore shown. B The activity of putative fluorescent tracers at FFA1 were assessed in Ca2+ mobilisation assays and compared to T360 (site two) and TUG-770 (site one) reference ligands. Data are mean ± SEM from N = 3 independent experiments and normalized against the response to T360. The ability of the three putative red tracers, TUG-2591 (C) TUG-2355 (D) and TUG-2287 (E) to produce a Ca2+ mobilisation responses are shown from cells expressing FFA1 tagged at its C (FFA1-Nluc) or N (Nluc-FFA1) terminal with Nluc or an untagged form of FFA1 (FFA1). Data in C are mean ± SEM from N = 3 (FFA1-Nluc, Nluc-FFA1) or N = 4 (FFA1), data in (D) are from N = 3, and data in (E) are from N = 2 independent experiments and normalized against the response to T360. Saturation BRET binding experiments compare specific BRET obtained from the C (purple, circles) or N (blue, squares) terminal FFA1 Nluc constructs with TUG-2591 (F), TUG-2355 (G), and TUG-2287 (H). Specific BRET was calculated by subtracting non-specific BRET binding obtained in the presence of the competing ligand, TUG-770 (10 μΜ). Saturation binding curve fit parameters with 95% confidence intervals are: TUG-2591- FFA1-Nluc, Kd = 23 (15−35) nM, BMax = 0.035 (0.032−0.038), Nluc-FFA1, Kd = 210 (150−300) nM, BMax = 0.31 (0.27-0.37); TUG-2355- FFA1-Nluc, Kd = 240 (75−810) nM, BMax = 0.022 (0.015−0.038), Nluc-FFA1, Kd 5 = 20 (260−1200) nM, BMax = 0.083 (0.060−0.14); TUG-2287- FFA1-Nluc, Kd = 25 (1.2−130) nM, BMax = 0.007 (0.005−0.01), Nluc-FFA1, Kd = 200 (120−340) nM, BMax = 0.11 (0.95−0.14). Saturation binding experiments (F−H) are mean ± SEM from N = 3 independent experiments. Source data are provided as a Source Data file.
