Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation

Abstract

The farnesoid X receptor (FXR), a member of the nuclear hormone receptor family, plays important roles in the regulation of bile acid and cholesterol homeostasis, glucose metabolism, and insulin sensitivity. There is intense interest in understanding the mechanisms of FXR regulation and in developing pharmaceutically suitable synthetic FXR ligands that might be used to treat metabolic syndrome. We report here the identification of a potent FXR agonist (MFA-1) and the elucidation of the structure of this ligand in ternary complex with the human receptor and a coactivator peptide fragment using x-ray crystallography at 1.9-A resolution. The steroid ring system of MFA-1 binds with its D ring-facing helix 12 (AF-2) in a manner reminiscent of hormone binding to classical steroid hormone receptors and the reverse of the pose adopted by naturally occurring bile acids when bound to FXR. This binding mode appears to be driven by the presence of a carboxylate on MFA-1 that is situated to make a salt-bridge interaction with an arginine residue in the FXR-binding pocket that is normally used to neutralize bound bile acids. Receptor activation by MFA-1 differs from that by bile acids in that it relies on direct interactions between the ligand and residues in helices 11 and 12 and only indirectly involves a protonated histidine that is part of the activation trigger. The structure of the FXR:MFA-1 complex differs significantly from that of the complex with a structurally distinct agonist, fexaramine, highlighting the inherent plasticity of the receptor.

Fig. 1.

FXR agonist ligands. The conventional steroid ring designations (A–D) are marked on MFA-1.

Fig. 2.

Results of titration studies to determine compound EC₅₀ values in a coactivator recruitment assay. The percentage maximal activity relative to a saturating concentration of CDCA (methods) is plotted as a function of ligand concentration. MFA-1 (open triangles), GW4064 (open circles), CDCA (solid triangles), MFA-2 (crosses), and MFA-3 (solid circles).

Fig. 3.

Overall structure of human FXR (blue) in complex with MFA-1 (yellow) and an SRC-1 coactivator peptide (magenta). Helices discussed in the text are labeled H1-H12, for reference.

Fig. 4.

General binding mode of MFA-1. (A) Final refined 2F_o−F_c electron density for MFA-1 contoured at 1.5 σ. (B) Results of the superposition of FXR molecules from the MFA-1(yellow) structure with rat FXR in complex with 6-ethyl-CDCA (magenta, PDB ID code 1OSV). The cα rmsd for the superposition is 1.177 Å. (C) Results of the superposition of FXR molecules from the MFA-1(yellow) structure with human ERβ in complex with estradiol (blue, PDB ID code 1QKT). The cα rmsd for the superposition is 4.217 Å.

Fig. 5.

Specific interactions between MFA-1 and FXR. (A) Interactions with the steroid ring system of MFA-1 (yellow). Side chains discussed in the text are colored cyan and labeled. (B) Stabilization of the helix 1–2 linker by MFA-1. Residues discussed in the text are labeled. (C) Stabilization of helix 12 by interactions with the hydroxy benzoyl group. Residues discussed in the text are colored cyan and labeled. Helix 3 is depicted as a thin trace for clarity.

Fig. 6.

Comparisons of the mechanism of receptor activation. (A) Alignment of the rat FXR (gray) structure in complex with 6-ethyl-CDCA (magenta) to the structure human FXR (blue) in complex with MFA-1 (yellow). Side chains discussed in the text are labeled and colored gray (rat FXR:6-ethyl CDCA), and blue (human FXR:MFA-1). Residue numbers in parentheses are for the rat receptor/6-ethyl-CDCA structure. (B) Alignment of the human FXR (gray) structure in complex with fexaramine (magenta, PDB ID code 1OSH) to the structure human FXR (blue) in complex with MFA-1 (yellow). Side chains discussed in the text are colored gray (human FXR:fexaramine), and blue (human FXR:MFA-1). Residue numbers in parentheses are for the fexaramine structure, and the cα rmsd for the superposition is 0.814 Å. Residue numbering differences arise from the use of different FXR isoforms that differ outside of the ligand binding domain contained in the crystal structures.