Fig. 4: Unique binding mode of the allosteric agonist compound 9n.

a The reported binding sites of PAMs in class A GPCRs. Site 1 (orange): LY2119620-bound M2R (PDB: 6OIK, iperoxo–LY2119620–M2R–Go), ML382-bound MRGPRX1 (PDB: 8DWG, BAM8-22–ML382-MRGPRX1-Gq), Site 2 (blue): MIPS521-bound A₁R (PDB: 7LD3, adenosine–MIPS521–A₁R–Gi2), Site 3 (pink): ZCZ011-bound CB1R (PDB: 7WV9, CP55940–ZCZ001–CB1-Gi), Cmpd-6FA-bound β2AR (PDB: 6N48, BI-167107–Cmpd-6FA–β2AR), Site 4 (purple): LY3154207-bound DRD1 (PDB: 7LJC, SKF-81297–LY3154207–DRD1–Gs). b Dose-response curves for the compound 9n-potentiated Niacin activation (left) and dose-response curves of Gi1 signaling induced by Niacin or compound 9n alone (right), error bars represent the standard deviation of curve fits from left (n = 5, 4, 6, 5, 5, and 6 top to bottom) right (n = 5, 6 top to bottom) independent experiments. c Detailed interactions between compound 9n and HCAR2. Hydrogen bonds are depicted as black dashed lines. d Effects of mutations in the compound 9n binding pocket on its potentiated Niacin activation. The Gi1 dissociation signal was detected by NanoBiT assay in the presence of 25 nM Niacin at EC20 concentration and increasing concentrations of compound 9n. Bars represent differences in calculated agonist potency (pEC50) for each mutant relative to the wild-type receptor (WT). Data are colored according to the extent of effect. ^nsP > 0.05; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparison test, compared with the response of WT, (the detailed P value for each condition is P  < 0.0001, P = 0.1983, P  < 0.0001, P = 0.9997, P < 0.0001, P < 0.0001, P = 0.5425, P = 0.0066, P = 0.0001, P < 0.0001, from left to right). Data are shown as the mean ± SEM from WT (n = 8), H184^5.34A (n = 3), T185^5.35A (n = 3), F186^5.36A (n = 4), Q187^5.37A (n = 3), E190^5.40A (n = 6), L194^5.44A (n = 3), L195^5.45A (n = 3), F198^5.48A (n = 6), F255^6.59A (n = 4), and L258^6.62A (n = 3) independent experiments). e Sequence alignment of compound 9n binding sites in the HCAR family. f PAM effect analysis of compound 9n in the HCAR family. Left to right: HCAR1: lactate; HCAR2: 3-HB; and HCAR3: 3-hydroxyoctanoic acid; ^nsP > 0.05; ^*P < 0.05; ^***P < 0.001; ^****P < 0.0001 (Data are the mean ± SEM from HCAR1-lactate (n = 3, 3), HCAR2-3-HB (n = 5, 3), and HCAR3-3-HC (n = 5, 3), independent experiments), Unpaired t test, two-tailed, with Welch’s correction or Mann–Whitney test, compared with the response of WT, the detailed P value for each condition is P > 0.9999, P = 0.0005, and P = 0.0347, from left to right). Source data are provided as a Source Data file.