Extended Data Fig. 4: Binding properties of FFA2 ago-PAMs at Site 1 and 2.

a, Loss of agonist activity and cooperativity with TUG-1375 for 4-CMTB at N²³⁰D FFA2, as measured by cAMP reduction assays. The data in the top row show that 4-CMTB lacks agonist action at N²³⁰D FFA2 whilst TUG-1375, compound 187 and AZ-1729 all activate this mutant as effectively as wild type. The data in the left and the middle panel of the bottom row indicate that 4-CMTB does not produce co-operativity with TUG-1375 at N²³⁰D FFA2 (left), while such effects of both AZ-1729 and compound 187 are retained at this mutant (middle). The data in the right panel indicate that the human FFA2 antagonist [³H]GLPG0974 displays high affinity binding to both wild type and N²³⁰D FFA2. Data are means +/– S.E.M. n = 3 (three biologically independent experiments). b, Structural comparison of FFA2 and FFA3 at Site 1. FFA2 and FFA3 are shown in green and pink, respectively. The space in FFA3 (middle panel, based on the structure PDB ID 8J21) corresponding to Site 1 in FFA2 (left panel) adopts a distinct conformation, which would result in a steric clash with 4-CMTB if it were to adopt a similar binding pose (right panel). c, Co-solvent simulations of AZ-1729 and compound 187. The middle figure shows the orthosteric ligand C4 and the probes as they are placed before the simulation, along with the cylinder (semi-transparent, blue) confining the probes around the area of interest, around ICL2. The top left figure shows AZ-1729 and the probe that allowed to identify its interaction with E106^3.49. The top right figure shows compound 187 and its probe molecule. The bottom left and right figures show the snapshots of the probe-AZ and probe-187, respectively, in MD simulations with the semi-transparent surface denoting the volume occupied by the probe for > 20% of the simulation time. d, Structural alignment of Site 2 for compound 187 in the structures with miniG_q and G_i. The interaction profile of compound 187 and the overall conformation of Site 2 including ICL2 are almost identical in these two structures. e, Effects of mutations in Site 2 on the potency of FFA2 modulators measured by [³⁵S]GTPγS binding assays. Data are means +/– s.e.m. for at least 3 experiments. pEC₅₀ and E_max values are listed in Extended Data Table 2. f, Undetectable β-arrestin-2 recruitment by AZ-1729 at Site 2 mutations in FFA2. β-arrestin-2 recruitment assays were performed with varying concentrations of TUG-1375 or AZ-1729 at wild type, E106G, and G102V FFA2. Data are means +/– s.e.m. n = 3 (three biologically independent experiments). Cmpd187 refers to compound 187.