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.
Subject(s)
Benzamides/pharmacology , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/chemistry , 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 , 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/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 TransductionABSTRACT
O-Alkylated quercetin analogs were synthesized and their anticancer activities were assessed by a high-throughout screening (HTS) method. The structure-activity relationships (SAR) showed that introduction of long alkyl chain such as propyl group at the C-3 OH position or short alkyl chain such as ethyl group at the C-4' OH position were very important for keeping inhibitory activities against the 16 cancer cell lines. Furthermore, when the two n-butyl groups were introduced into the C-3, C-7 or C-4', C-7 positions, the anticancer activity was enhanced.
Subject(s)
Antineoplastic Agents/pharmacology , Quercetin/pharmacology , Alkylation , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HeLa Cells , High-Throughput Screening Assays , Humans , Molecular Structure , Quercetin/chemical synthesis , Quercetin/chemistry , Structure-Activity RelationshipABSTRACT
Using a high-throughout screening approach, the anticancer activities of 16 O-methylated (OMe) analogs of quercetin were assessed. The structure-activity relationships showed that OMe moieties at the 4' and/or 7 positions were important for maintaining inhibitory activities against the 16 cancer cell lines. Furthermore, when the OH groups at the 3' and 4' positions were both replaced by OMe moieties, anticancer activity was enhanced.