Figure 1. Chemical Structures of Identified and Known FXR Ligands and Their In Vitro Binding Affinities.
(A) A schematic diagram of the ligand discovery phase and characterization scheme of identified high-affinity agonists. (B) Selected regions of interest for SAR evaluation of prototypical structure lead compounds 1. Region I, right-hand aromatic system; Region II, Acyl group region; Region III, left-hand benzopyran ring system. Compound 2 was produced by systematic optimization of Regions I and II. Fexaramine was selected from a final 94 membered combinatorial library of Region III. (C) Structures of lead compounds (and their EC50 values in a cell-based assay) selected for further biological evaluation as FXR agonists. A, fexaramine (EC50 = 25 nM); B, fexarine (EC50 = 38 nM); C, fexarene (EC50 = 36 nM); D, SRI-1 (EC50 = 377 nM); E, SRI-2 (EC50 = 343 nM). The identified compounds are structurally distinct from known FXR agonists F, CDCA a physiological low-affinity ligand, and the high-affinity ligand G, GW4064 (EC50 = 80 nM). (D) Identified compounds fexaramine, fexarine, fexarene, SRI-1, and SRI-2 are agonists for FXR in vitro. A FRET-based ligand binding assay was carried out in agonist mode using GW4064 as the control ligand. Increasing amounts of the compounds were added as indicated. Binding reactions contained 8 nM europium-labeled GST-FXR-LBD fusion protein and 16 nM allophycocyanin-labeled SRC-1 receptor binding peptide. Results are expressed at 1000*(665 nm/615 nm).