Studies On The Discovery Of New Drugs Targeting HD-NRs

The discovery and identification of novel drug targets for preventing and treating diseases is the purpose of the work for all researchers worldwide.Nuclear receptors(NRs) are a superfamily of ligand-activated transcription factors that play critical role in many biological processes including embryonic growth and development,cell differentiation and apoptosis,immune respond and metabolic homeostasis.Heterodimer nuclear receptors are the most important members of NR,The Retinoid Acid receptors(RARs),the liver X receptors(LXRs) and the farnesoid X receptor(FXR) are belonged to heterodimer nuclear receptor subfamily.The metabolism of retinoids,glucose,fat,cholesterol and bile acids is controlled,in part,by a subset of heterodimer nuclear receptors including RARs, LXRs and FXR.Studies In vitro demonstrated that they regulate genes transcription through binding to specific DNA respond elements with Retinoid X receptor(RXRs),which may involved in numerous diseases,including Cardio-cerebral-vasculardiseases,type2diabetes,obesity,skindisorder, inflammation,neurodegenerative disease and cancer.They may be one kind of potential novel targets in drug discovery for metabolism disorder disease.The purpose of our study was to evaluate heterodimer NR as new drug targets,set up the screening platform,and finally find the active new lead compounds.Our research would be presented as followed.PartⅠAs a classic heterodimer NR,the RARs have been associated with several diseases such as cancer or skin disorders on the basis of epidemiological,clinical, and experimental investigations in human and animals.The RAR agonists,retinoids are successfully used in a variety of chemopreventive and chemotherapeutic settings.However,some of the traditional RAR agonists,such as all trans retinoic acid,had unexpected toxic and side-effect.Exploring the higher potential,lower toxic RAR agonist is still important in drug research,a stable,sensitive,reproducible High content screening(HCS) and evaluation platform for searching RAR specific agonists was successfully established With the help of virtual screening approach,the mode of drug discovery to NR was explored and utilized to screen new leading compounds from 20,0000 compounds.Compound 7672 and 7666 were determined.The compounds 7672 show a much better activity to RAR than positive compound 9-cis-retinoid acid(9-CRA) on HCS.The further mechanism evaluation to 7672 was complished.PartⅡMammalian one-hybrid system is a functional transactivation cell-based assay utilized for detecting ligands/nuclear receptor interactions.This assay system allows one to detect ligands/NR interactions quantitatively by reporter gene expression and also helps us to determine the potential biological function of a compound in a certain cellular background.In addition,mammalian one-hybrid system has one major advantage that can eliminate the interference factors of mammal cell endogenous nuclear receptors for using the yeast GAL4 response element in mammalian cells.To discover novel lead compounds for metabolic disorder diseases targeting heterodimer NRs,based on the mammalian one-hybrid technology,a HTS and activity evaluation platform including LXRαand FXR was developed.The LBDs of LXRa and FXR were amplified from the mRNA of liver by RT-PCR,and fused to the DNA binding domain(DBD) of yeast GAL4 of plasmid pBIND to construct chimera expression plasmid.The pGL5 was a reporter plasmid contain Five copies of the GAL4 DNA binding site,which was inserted into upstream of the promoter.The cell-based HTS assays were evaluated by positive compounds after a serial of cotransfection condition optimization.All the positive compounds had the transactivities in dose-dependent manner.The assays were proved to be robust and reproducible as suggested by high S/N value and Z' factors(0.5-0.7)in 96-well format.In the process of screening,4 active compounds were screened from pure natural compounds library.They were proved to be a selective LXR agonist, although show weak activation to FXR.In addition,reporter and expression plasmid were constructed to establish heterodimer HCS platform.and then evaluated through transfection.to mammalian cells.PartⅢUpon agonist binding,nuclear receptors undergo a conformational change that increases their affinity for coactivators.Recruitment of coactivator to agonist-bound nuclear receptor is a critical step in the formation of an active transcription complex on DNA and then induced target gene transcription.. Studies have demonstrated that coactivator fragments containing the motif LXXLL,where L is leucine and X is any amino acid,can bind to nuclear receptors in an agonist-dependent manner.Interaction between the LXRa LBD(or FXR) and a short peptide contain the motif from the coactivator SRC-1,measured by fluorescence polarization can provides an indication of ligand binding and receptor activation.In this study,we synthesized a short synthetic FITC-labeled peptide containing an LXXLL motif and used it to develop a fluorescence polarization assay for agonist binding to LXR or FXR.In this homogeneous biochemical assay,the greater the extent of FITC labeled peptide binding to NR, the greater the extent of fluorescence polarization observed.The FP-based assay can be used to identify the binding of test compounds to all metabolic heterodimer nuclear receptors,including RAR,CAR,VDR,PPAR,LXR,FXR et al.In the process of FP screening in vitro,4 active compounds were proved not only to activating LXR,but they could.bind directly to LXR and recruit coactivator to agonist-bound nuclear receptor. This HTS and activity evaluation platform including cell-based and in vitro FP assay targeting nuclear receptors and active compounds obtained by this platform lay basis for drug development of metabolic disorder diseases at the early stage of the drug discovery process and detailed understanding of the molecular basis for the biology of these nuclear receptors.PartⅣActive compounds,Reveratrol tetramers were found to be selective LXR agonist in this study,and for the first time it was revealed that they play role in anti-atherosclerosis through LXR pathway.We studied the effects of active compounds on the expression of gene associated with HDL cholesterol metabolism and reverse cholesterol transport.We found active compounds up-regulate the mRNA expression of LXR target genes,and showed the effects on inhibition of foam cell formation and promotion cholesterol efflux from oxLDL loaded macrophage.They also had the effects on the anti-inflammation by repression of the inflammatory cytokines and inhibiting ox-LDL induced adhesion of monocytes to t endothelial cell line.They also be a good anti-oxidant by increasing SOD activity and reducing ox-LDL induced MDA production.In summary,the present study focused on the key steps for new drugs discovery,such as drug target validation,high-throughput screening and hits activities evaluation.Our study would promote the new drugs discovery for preventing and treating metabolism disorder diseases.