scholarly journals Characterization of a stable cell line expressing human Na + /taurocholate cotransporting polypeptide for high throughput screening

2009 ◽  
Vol 23 (S1) ◽  
Author(s):  
Megan Roth ◽  
Yazen Alnouti ◽  
Bruno Hagenbuch
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Stable Cell Line

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.

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.

scholarly journals Inducible APOBEC3G-Vif Double Stable Cell Line as a High-Throughput Screening Platform To Identify Antiviral Compounds

2009 ◽  
Vol 54 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Boris Nowotny ◽  
Thomas Schneider ◽  
Gabriele Pradel ◽  
Tanja Schirmeister ◽  
Axel Rethwilm ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Yellow Fluorescent Protein ◽  
26S Proteasome ◽  
Stable Cell Line ◽  
Antiviral Compound ◽  
Compound Libraries ◽  
Antiviral Compounds

ABSTRACT Inhibition of the interaction of the human cytidine-deaminase APOBEC3G (A3G) with the human immunodeficiency virus (HIV) type 1-specific viral infectivity factor (Vif) represents a novel therapeutic approach in which a cellular factor with potent antiviral activity (A3G) plays a key role. In HIV-infected cells, the interaction of Vif with A3G leads to the subsequent degradation of A3G by the 26S proteasome via the ubiquitin pathway and to the loss of antiviral activity. To establish a stable and convenient cellular testing platform for the high-throughput screening of potential antiviral compound libraries, we engineered a double transgenic cell line constitutively expressing an enhanced yellow fluorescent protein expressor (EYFP-A3G) fusion as well as a Tet-Off controllable Vif protein. With this cell line, we were able to measure precisely the Vif-induced degradation of A3G in the presence of potential antiviral compounds in an easy-to-handle, robust, and practical high-throughput multiwell plate format with an excellent screening window coefficient (Z factor) of 0.67.

scholarly journals Identification of a juvenile-hormone signaling inhibitor via high-throughput screening of a chemical library

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takumi Kayukawa ◽  
Kenjiro Furuta ◽  
Keisuke Nagamine ◽  
Tetsuro Shinoda ◽  
Kiyoaki Yonesu ◽  
...  
Keyword(s):  
Juvenile Hormone ◽  
Cell Line ◽  
Signaling Pathway ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Ex Vivo ◽  
Agricultural Field ◽  
Chemical Library ◽  
Field Development

Abstract Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins

2019 ◽  
Vol 60 (5) ◽  
pp. 1082-1097 ◽  
Author(s):  
Panneerselvam Krishnamurthy ◽  
Yukiko Fujisawa ◽  
Yuya Takahashi ◽  
Hanako Abe ◽  
Kentaro Yamane ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
High Density ◽  
Biosynthesis Pathway ◽  
Mutant Library ◽  
Triterpenoid Saponins

scholarly journals A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

2013 ◽  
Vol 58 (1) ◽  
pp. 110-119 ◽  
Author(s):  
Chi-Chen Yang ◽  
Han-Shu Hu ◽  
Ren-Huang Wu ◽  
Szu-Huei Wu ◽  
Shiow-Ju Lee ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitor ◽  
Current Treatment ◽  
Stable Cell Line ◽  
Epidemic Disease ◽  
Molecule Inhibitor ◽  
Ns3 Protease

ABSTRACTDengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC50) of 1.03 ± 0.09 μM. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future.

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