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-Å 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.NR1H4 ͉ bile acid receptor ͉ nuclear receptor ͉ x-ray crystallography T he farnesoid X receptor (FXR) plays key roles in regulating cholesterol and bile acid homeostasis (1-4). Central to this function is the ability of bile acid-activated FXR to downregulate the expression of Cyp7a (1, 5, 6), the rate-limiting step in the liver for the conversion of free cholesterol to bile acids, and the up-regulation of the bile salt excretion pump (BSEP), which functions to pump excess bile acids into the gall bladder for eventual fecal excretion (7). Treatment of ob/ob and db/db mice with the synthetic FXR agonist GW4064 significantly improves hypercholesterolemia (8) and lowers free fatty acids (9, 10) and triglyceride levels (11). Additional research has shown that fxr Ϫ/Ϫ mice show insulin resistance and impaired glucose tolerance (8,10,12), and that expression of constitutively active FXR in the liver results in hypoglycemia (10). Similarly, treatment with GW4064 increases insulin sensitivity in ob/ob and db/db mice (8, 10). As such, FXR agonists may have utility in treating metabolic syndrome, a clustering of cardiovascular risk factors characterized by dyslipidemia (elevated triglyceride and low HDL levels), insulin resistance, and poor glucose regulation. Several excellent reviews have summarized the current state of FXR understanding (13,14) and help build the case for the development of FXR modulators for the treatment of diabetes and metabolic syndrome (15).FXR belongs to the lar...

Bile acid receptors as targets for the treatment of dyslipidemia and cardiovascular disease

“…Surprisingly, different gene profi les are induced by fexaramine, CDCA, or GW4064 in mouse hepatocytes ( 122 ). The conformation of the ligand-receptor complex of FXR with fexaramine differs from that with CDCA or MFA-1, a synthetic steroidal FXR agonist ( 122,123 ), which might be linked to the differential induction of target genes by the respective ligands (SBARM concept).…”

“…Structurally different ligands can bind differently to the FXR ligand binding pocket, whose intrinsic plasticity is defi ned by two cavities that can fi t ligand substructures ( 130,131 ). Depending on the (stereo)chemical properties of the ligand, the exact site of binding, the closeness of the ligand-receptor conformation, and the strength of the binding can differ ( 122,123,131 ). The specifi c ligand-receptor conformation can determine the ability of coregulators to act (i.e., activators to bind or repressors to dissociate), which defi nes the gene selectivity of a given ligand.…”

Exploring the binding diversity of intrinsically disordered proteins involved in one‐to‐many binding

“…In summary, NCOA binding molecules include many kinds of nuclear receptors, including androgen receptor, estrogen receptor, nuclear receptor subfamily 1, group I member 3 (NR1I3/CAR), bile acid receptor, and pregnane X receptor. [57][58][59][60][61] Other detailed investigation into the MoRFs with similar-fold partners was performed and discussed in our previous work. 52 …”

Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor

“…To date increasing numbers of FXR␣ agonists have been discovered, such as GW4064, fexaramine, 6a-ethyl-CDCA, MFA-1, XL335, and ivermectin (7)(8)(9)(10)(11)(12). However, it was determined that FXR␣ activation by full agonist may also cause undesired side effects in animals.…”