Establishment And Application Of A High-throughput Drug Screening Platform For Liver X Receptor Agonists

Atherosclerosis is the leading cause of mortality in developed countries, and increased rapidly in developing countries in recent years. Lots of studies demonstrate that Liver X receptors (LXRs) plays an important role in development of atherosclerosis. To date, LXRs has been confirmed to be the promising anti-atherogenic pharmacological target.LXRs belong to a family of nuclear receptors. LXRs were initially described as orphan receptors and oxidized cholesterol derivatives (oxysterols) were later identified as their natural ligands. LXRs were heterodimerized with retinoid X receptors to modulate the transcription of the target genes by binding to the DNA LXR response element (LXRE). LXRs serve as cholesterol sensors that regulate the expression of multiple genes involved in the efflux, transport, and excretion of cholesterol. In the recent years, LXRs have been characterized as key transcriptional regulators of lipid and carbohydrate metabolism. In addition, several studies confirmed the role for LXRs agonists in the control of both inflammation and cholesterol homeostasis in the brain and central nervous system (CNS). Therefore, the ability of LXRs that integrate metabolic and inflammatory signaling makes them particularly attractive targets for intervention in human atherosclerosis, hypercholesterolemia and typeⅡdiabetes.High throughput screening (HTS) is a new method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing and control software, liquid handling devices, and sensitive detectors, HTS allows a researcher to quickly conduct millions of tests. Through this process one can rapidly identify active compounds which modulate a particular physiology pathway.To discover novel lead compounds for atherosclerosis, based on the reportor gene assays, a high throughput screening (HTS) platform for identification of LXRs agonists was developed in present study.In this study, we constructed four different high throughput screening reportor gene assays for LXRs agonists. The cDNA encoding the LXRαand LXRβwas amplified by reverse transcription PCR from the liver cell total mRNA, and fused to the vector of pBIND, pTARGET to construct chimera expression plasmid pBIND-LXRβ,pTARGET-LXRβ. The ABCA1 promoter was amplified by PCR using human genomic DNA (extracted from the cultured U937 cells), and fused it to the vector of pGL3 vector to construct reporter plasmid pGL3-ABCA1 (promoter)-Luc. Four copies of the LXRE were synthezied and inserted into the pGL3 vector to construct a reporter plasmid pGL3-4×LXRE-Luc.Four stable and sensitive cell-based high- throughput screening model for LXRs agonists were established by cotransfection of pGL3-GAL4-Luc with pBIND-LXRa or pBIND-LXRβ, or pTARGET- LXRβwith pGL3-ABCA1 (promoter)-Luc or pGL3-4×LXRE -Luc. TO901317, a LXRs agonist, induced the expression of luciferase gene in a dose-dependent manner with high Signal/Blank ratio (S/B). After optimization, these assay produced high S/B values (3.47-6.16), acceptable Z’-factor values (0.42-0.71) in 96-well format. The EC50 values of TO901317 were about 8.9μM-19.7μM.Using these assay, seven compounds, Asperazine,3’,4’,5’,7’-tetrahydroxy-8-methoxy isoflavone, Herbimycin A, Oxepinamide D, Genistein, BY-16 and H-3, were identified as positive compounds that potently active the LXRs.These active compounds were further comfirmed as regulators of LXRs via analyzing the expression of LXRs target genes by real-time PCR. In addition, these compounds could inhibit NO production in macrophage and showed low cytotoxicity to cell membrane, These studies suggest that these compounds may have potential effects on anti-atherosclerosis and anti-inflammatory.In conclusion, a HTS platform for identification and evaluation of upregulators of LXRs was established in this work, and seven active compounds were identified as LXRs regulators using this screening platform. These compounds are expected to have the potential to prevent atherosclerotic diseases and the other related diseases.