Fig. 1: Development of TUG-2597, a green fluorescent tracer binding to FFA1 site two.

A. Chemical structures of putative site two fluorescent tracers based on Cpd 6h. The linkers used and the position of an NBD green fluorophore are shown. B The activity of putative fluorescent tracers at FFA1 were assessed in Ca²⁺ mobilisation assays and compared to FFA1 site two (Cpd 6h) and site one (TUG-770) reference ligands. Data are mean ± SEM from N = 3 independent experiments and normalized against the response to T360. C The proximity between the NBD tag of TUG-2597 (blue spheres) and Nluc is predicted to be sufficient for BRET to occur both with the Nluc tagged either at the C (left) or N (right) terminal of FFA1, shown with overlapping green and blue spheres in the illustration. A site one ligand is shown with red spheres. D Comparison of Ca²⁺ responses to TUG-2597 in cells expressing FFA1 tagged at its N (Nluc-FFA1) or C (FFA1-Nluc) terminal with Nluc, to cells expressing an untagged form of FFA1 (FFA1). Data are mean ± SEM from N = 3 (FFA1-Nluc, Nluc-FFA1) or N = 4 (FFA4) independent experiments and normalized against the response to T360. Specific BRET binding curve for TUG-2597 obtained in cells expressing an FFA1 construct tagged at its C (E) or N (F) terminal with Nluc are shown. Specific BRET was determined by subtracting non-specific BRET binding obtained in the presence of the competing ligand, T360 (10 μM). Data are expressed as a percentage of the maximal signal. G Saturation BRET binding curves for TUG-2597 are compared between the C terminal and N terminal FFA1 constructs. Data are shown as the BRET ratio (540 nm / 450 nm emission). Saturation binding experiments (E−G) show mean ± SEM from N = 3 independent experiments. Source data are provided as a Source Data file.