scholarly journals Identification of Novel Human High-Density Lipoprotein Receptor Up-regulators Using a Cell-Based High-Throughput Screening Assay

2007 ◽  
Vol 12 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Yuan Yang ◽  
Zhongbing Zhang ◽  
Wei Jiang ◽  
Lei Gao ◽  
Guiyu Zhao ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Cell Line ◽  
High Throughput ◽  
Hepg2 Cells ◽  
High Throughput Screening ◽  
Density Lipoprotein ◽  
High Density ◽  
Luciferase Reporter ◽  
Stable Cell Line ◽  
Active Compounds

Scavenger receptor class B type I (SR-BI) is the high-affinity high-density lipoprotein (HDL) receptor, and CLA-1 is the human homologue of the murine SR-BI. CLA-1/SR-BI receptor has been suggested as a new preventative and/or therapeutic target for atherosclerosis due to its pivotal role in overall HDL cholesterol (HDL-C) metabolism and its antiatherogenic activity in vivo. To search for active compounds that can increase CLA-1 transcription, a novel cell-based assay was developed for application in high-throughput screening (HTS). Human hepatoma HepG2 cells were transfected with a CLA-1-promoter-luciferase reporter gene construct, and the stable transfected cell line was selected and named CLAp-LUC HepG2. With rosiglitazone as a positive control, this stable cell line was used to establish a specific CLA-1 gene expression assay in a 96-well microplate format. The evaluating parameter Z' value of 0.64 showed that this cell-based HTS assay was robust and reliable. Screening of 6000 microbial secondary metabolite crude extracts identified 8 positive strains. Between 2 identified CLA-1 up-regulators produced by actinomycete strain 04-4776, 4776B may stimulate not only the expression of CLA-1 on the transcriptional and translational levels but also the activity of CLA-1 to uptake the HDL-C in HepG2 cells. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new antiatherosclerosis agents.

Apolipoproteins C-II and C-III inhibit selective uptake of low- and high-density lipoprotein cholesteryl esters in HepG2 cells

2005 ◽  
Vol 37 (6) ◽  
pp. 1308-1318 ◽  
Author(s):  
Karine Huard ◽  
Philippe Bourgeois ◽  
David Rhainds ◽  
Louise Falstrault ◽  
Jeffrey S. Cohn ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Hepg2 Cells ◽  
Density Lipoprotein ◽  
High Density ◽  
Cholesteryl Esters ◽  
Selective Uptake

scholarly journals A High-Throughput Screening Platform Targeting PDLIM5 for Pulmonary Hypertension

2016 ◽  
Vol 21 (4) ◽  
pp. 333-341 ◽  
Author(s):  
Han Cheng ◽  
Tianji Chen ◽  
Merve Tor ◽  
Deborah Park ◽  
Qiyuan Zhou ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Drug Discovery ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Pdz Domain ◽  
Luciferase Reporter ◽  
Targeted Drug ◽  
Screening Platform ◽  
Targeted Drug Discovery

Pulmonary arterial hypertension is a complex disease with multiple etiologic factors. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and three LIM domains at its C-terminus. We have previously shown that overexpression of PDLIM5 prevents hypoxia-induced pulmonary hypertension (PH), and deletion of PDLIM5 in smooth muscle cells enhances hypoxia-induced PH in vivo. These results suggest that PDLIM5 may be a novel therapeutic target of PH. In this study, we aim to establish a high-throughput screening platform for PDLIM5-targeted drug discovery. We generated a stable mink lung epithelial cell line (MLEC) containing a transforming growth factor–β/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We found that in MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, Smad3, and pSmad3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1200 compounds) and identified and validated paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells. Our study suggests that this system is robust and suitable for PDLIM5-targeted drug discovery.

Low- and High-Density Lipoprotein Metabolism in HepG2 Cells Expressing Various Levels of Apolipoprotein E†

Biochemistry ◽  
2000 ◽  
Vol 39 (51) ◽  
pp. 16084-16091 ◽  
Author(s):  
Daniel Charpentier ◽  
Caroline Tremblay ◽  
Eric Rassart ◽  
David Rhainds ◽  
Anick Auger ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Apolipoprotein E ◽  
Hepg2 Cells ◽  
Lipoprotein Metabolism ◽  
Density Lipoprotein ◽  
High Density

scholarly journals Development of a Cell-Based High-Throughput Screening Assay to Identify Porcine Host Defense Peptide-Inducing Compounds

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Zhuo Deng ◽  
Jing Wang ◽  
Wentao Lyu ◽  
Xuwen Wieneke ◽  
Robert Matts ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Hdac Inhibitors ◽  
Luciferase Reporter ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
A Cell

Novel alternatives to antibiotics are needed for the swine industry, given increasing restrictions on subtherapeutic use of antibiotics. Augmenting the synthesis of endogenous host defense peptides (HDPs) has emerged as a promising antibiotic-alternative approach to disease control and prevention. To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1 kb porcine β-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1–5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24 h exposure to HDAC inhibitors. In conclusion, a cell-based high-throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

scholarly journals Potential High-Throughput Assay for Screening Inhibitors of West Nile Virus Replication

2003 ◽  
Vol 77 (23) ◽  
pp. 12901-12906 ◽  
Author(s):  
Michael K. Lo ◽  
Mark Tilgner ◽  
Pei-Yong Shi
Keyword(s):  
West Nile Virus ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Health Priorities ◽  
Stable Cell Line ◽  
West Nile ◽  
Subgenomic Replicon ◽  
Genes Encoding ◽  
High Throughput Assay

ABSTRACT Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encoding Renilla luciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.

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