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

Xing Xu; Xin Xu; Peng Liu; Zhi-yuan Zhu; Jing Chen; Hai-an Fu; Li-li Chen; Li-hong Hu; Xu Shen

doi:10.1074/jbc.M114.630475

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

J Biol Chem. 2015 Aug 7;290(32):19888-99. doi: 10.1074/jbc.M114.630475. Epub 2015 Jun 22.

Authors

Xing Xu¹, Xin Xu², Peng Liu², Zhi-yuan Zhu², Jing Chen², Hai-an Fu³, Li-li Chen⁴, Li-hong Hu⁵, Xu Shen⁶

Affiliations

¹ From the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China, Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Shanghai Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
² From the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China.
³ Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
⁴ From the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China, lilichen@simm.ac.cn.
⁵ From the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China, lhhu@simm.ac.cn.
⁶ From the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China, xshen@mail.shcnc.ac.cn.

Abstract

Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research.

Keywords: FXR; antagonist; crystal structure; homodimerization; metabolic disease; molecular pharmacology; nuclear receptor; transcription target gene.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

ATP Binding Cassette Transporter, Subfamily B, Member 11
ATP-Binding Cassette Transporters / antagonists & inhibitors
ATP-Binding Cassette Transporters / genetics
ATP-Binding Cassette Transporters / metabolism
Animals
Benzamides / chemistry
Benzamides / pharmacology*
Crystallography, X-Ray
Gene Expression Regulation
Glucose-6-Phosphatase / antagonists & inhibitors
Glucose-6-Phosphatase / genetics
Glucose-6-Phosphatase / metabolism
Hep G2 Cells
Hepatocytes / cytology
Hepatocytes / drug effects
Hepatocytes / metabolism
Humans
Isoxazoles / antagonists & inhibitors
Isoxazoles / pharmacology
Male
Mice
Mice, Knockout
Molecular Docking Simulation
Mutation
Phosphoenolpyruvate Carboxykinase (ATP) / antagonists & inhibitors
Phosphoenolpyruvate Carboxykinase (ATP) / genetics
Phosphoenolpyruvate Carboxykinase (ATP) / metabolism
Primary Cell Culture
Protein Isoforms / agonists
Protein Isoforms / antagonists & inhibitors
Protein Isoforms / genetics
Protein Isoforms / metabolism
Receptors, Cytoplasmic and Nuclear / agonists
Receptors, Cytoplasmic and Nuclear / antagonists & inhibitors*
Receptors, Cytoplasmic and Nuclear / chemistry*
Receptors, Cytoplasmic and Nuclear / genetics
Receptors, Cytoplasmic and Nuclear / metabolism
Receptors, Leptin / deficiency
Receptors, Leptin / genetics
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Retinoid X Receptors / agonists
Retinoid X Receptors / genetics
Retinoid X Receptors / metabolism
Signal Transduction

Substances

ATP Binding Cassette Transporter, Subfamily B, Member 11
ATP-Binding Cassette Transporters
Abcb11 protein, mouse
Benzamides
Isoxazoles
Protein Isoforms
Receptors, Cytoplasmic and Nuclear
Receptors, Leptin
Recombinant Proteins
Retinoid X Receptors
leptin receptor, mouse
nuclear receptor subfamily 0, group B, member 2
farnesoid X-activated receptor
Glucose-6-Phosphatase
Phosphoenolpyruvate Carboxykinase (ATP)
GW 4064

Associated data

PDB/3DCT
PDB/4OIV