The Structural Mechanism Of Antihypertensive 1,4-Dihydropyridine Drugs In Protecting The Liver By Targeting The Nuclear Receptor FXR

Farnesoid X receptor(FXR,NR1H4)plays an important role in regulating bile acid synthesis and cholesterol homeostasis.Despite the considerable attention of FXR as a drug target in metabolic diseases,the therapeutic potentials and research significance of FXR selective ligands remain to be further studied.In this study,we elucidated the molecular mechanism and structure-activity relationship of antihypertensive drugs targeting FXR in regulating hepatic metabolism.We reveal and identify a series of antihypertensive 1,4-dihydropyridine(DHPs)as FXR ligands using a high-throughput FDA compound library screening and show that they bind to FXR to induce different conformational changes,thereby regulating FXR properties in recruiting coregulato including Steroid receptor coactivator SRC2-3 and the transcriptional activity of the FXR.In order to further study the molecular mechanism of different DHPs binding to FXR,the crystal structures of FXR/SRC2-3/DHPs were analyzed and reported for the first time.The crystal structures of the complex reveal unique binding mode of DHPs binding in the FXR ligand binding pocket including a three-blade propeller shape for the space occupied by the DHPs molecules embedded in the proper cavities.DHPs were mainly located between H7 and H11 to form a small binding cavity to regulate the binding flexibility of DHPs,which is distinct from known ligands Chenodeoxycholic acid(CDCA)and GW4064.Site-directed mutagenic analyses further showed distinct results for the activation of FXR by the four DHPs in the structure-activity relationship,highlighting the flexibility of FXR ligand binding pocket and the differential roles of these mutagenic residues in recognizing various DHPs and regulating FXR functions.Treatment of wild-type mice,but not of FXR-null mice,with Cilnidipine significantly from acetaminophen(APAP)-induced actue injury reduced serum Alanine aminotransferase ALT,Aspartate aminotransferase AST,Lactate dehydrogenase LDH levels and increased hepatic Glutathione S-transferase GSH levels,suggesting that cilnidipine regulates biomarkers of hepatic injury through FXR.Notably,the cilnidipine pre-treatment efficaciously protected the liver of WT mice from injury,but not FXR-null mice,suggesting that cilnidipine improves liver injury through FXR.Likewise,TUNEL staining further showed obvious apoptosis characteristics in the liver sections of APAP-induced hepatocyte death,cilnidipine pre-treatment significantly reduced the extent of TUNEL-positive apoptosis areas in WT mice than FXR KO controls,suggesting that FXR is required for the protective role of cilnidipine in APAP-induced hepatotoxicity and damage.To further assess the molecular mechanisms underlying the hepatoprotective effects of cilnidipine,we then measured the mRNA levels of related genes in the liver tissues of APAP-treated WT and FXR KO mice.The mRNA levels of genes Glutamate cysteine ligase Glutathione s-transferase a3 Gsta3,Glutathione peroxidase 1 Gpxland Bile salt export pump BSEP were all significantly increased in clinidipine pre-treated WT mice,but not FXR KO mice.It is considered that cilnidipine likely exerts its hepatoprotective effects by activating FXR and subsequently upregulating the expression of a number of genes involved in xenobiotic metabolism.These results reaffirm that cilnidipine regulates gene expression and hepatoprotective effects in an FXR-dependent manner.Considering that DHPs are widely used clinical anti-hypertensive drugs,our findings show that DHPs are not only provides valuable insight into the design and development of more beneficial next-generation,anti-hypertensive Ca2+ blocker drugs,but also reveal an alternative safe and effective structural template for the design of FXR ligands with therapeutic potentials to rapid clinical applications.Moreover,based on the established drug-target relationship,DHPs may potentially be used to treat FXR-mediated related diseases other than hypertension,and our research also provides a powerful screening strategy for further exploring new functions of "old drugs".