Fig. 1

Ligand binding and functional assays of compound 1. a Competitive radioligand binding for compound 1. GPR40 membranes (prepared from stably transfected cells) were incubated with either [³H]-TAK-875 (triangles), n = 3, or [³H]-AM-1638 (circles), n = 4, in the presence of unlabeled compound 1, as described in the Methods section. Percent specific binding (y-axis) was plotted against the log concentration of compound 1 (x-axis) and fit with a four parameter nonlinear logistic curve function with variable slope (GraphPad Prism, v7.00). The K_i for compound 1 in [³H]-AM-1638 binding was 0.81 nM. An inhibition curve for [³H]-TAK-875 binding was not drawn since the addition of compound 1 resulted in enhanced binding activity. Data points represent the mean of single concentration determinations of independent experiments performed on different days with the same stock sample of compound. Error bars indicate the s.e.m. The affinity modulation factor (α) was calculated using the allosteric modulator titration equation within GraphPad Prism. b Ca²⁺ mobilization assay (FLIPR^®) using stably transfected cells. GPR40- Gα_q signaling by compound 1 (triangles), n = 4. compound 1 acts as a full agonist relative to 500 µM of the natural ligand, linoleic acid. (EC₅₀ ~72 nM; Top = 136.3%; error bars indicate s.e.m.) c cAMP accumulation assay using stably transfected cells. Level of cAMP is measured as relative luminescence units (RLU). GPR40-Gα_s signaling by compound 1 (diamonds), n = 6. (EC₅₀ ~125.8 nM; error bars indicate s.e.m.)