Identification of Upregulators of Human ATP-Binding Cassette Transporter A1 via High-Throughput Screening of a Synthetic and Natural Compound Library

2008 ◽  
Vol 13 (7) ◽  
pp. 648-656 ◽  
Author(s):  
Jie Gao ◽  
Yanni Xu ◽  
Yuan Yang ◽  
Yi Yang ◽  
Zhihui Zheng ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Natural Compound ◽  
Oncostatin M ◽  
Assay Method ◽  
Atp Binding ◽  
Compound Library ◽  
Atp Binding Cassette Transporter ◽  
High Throughput Screening Assay ◽  
Atp Binding Cassette

The ATP-binding cassette transporter A1 (ABCA1) is a membrane transporter that directly contributes to high-density lipoprotein (HDL) biogenesis by mediating the cellular efflux of cholesterol and phospholipids to lipid-poor apolipoprotein A-I. Therefore, identification of a novel upregulator of ABCA1 would be beneficial for atherosclerosis prevention and/or therapy because of its pivotal role in cholesterol homeostasis and HDL metabolism. In this study, a high-throughput assay method for ABCA1 upregulators was developed and used for screening a synthetic and natural compound library. The cell-based high-throughput screen is conducted in a 96-well format using the human hepatoma HepG2 cells stably transfected with ABCA1 promoter-luciferase construct and calibrated with reference ABCA1 upregulators (oxysterols, 9-cis-retinoic acid, thiazolidinediones, cyclic adenosine monophosphate, verapamil, fenofibrate, and oncostatin M). Among 2600 compounds, 4 microbial compounds (pyrromycin, aclarubicin, daidzein, and pratensein) were picked up as hits by the high-throughput screening assay, and those compounds were further identified as upregulators of ABCA1 expression by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. ( Journal of Biomolecular Screening 2008:648-656)

scholarly journals New High-Throughput Screening Assay To Reveal Similarities and Differences in Inhibitory Sensitivities of Multidrug ATP-Binding Cassette Transporters

2009 ◽  
Vol 53 (4) ◽  
pp. 1516-1527 ◽  
Author(s):  
Marcin Kolaczkowski ◽  
Anna Kolaczkowska ◽  
Noboru Motohashi ◽  
Krystyna Michalak
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Plasma Membranes ◽  
Dynamic Range ◽  
Genetic Manipulation ◽  
High Sensitivity ◽  
Atp Binding ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Atp Binding Cassette

ABSTRACT Cdr1p is the major ATP-binding cassette multidrug transporter conferring resistance to azoles and other antifungals in Candida albicans. In this study, the identification of new Cdr1p inhibitors by use of a newly developed high-throughput fluorescence-based assay is reported. The assay also allowed monitoring of the activity and inhibition of the related transporters Pdr5p and Snq2p of Saccharomyces cerevisiae, which made it possible to compare its performance with those of previously established procedures. A high sensitivity, resulting from a wide dynamic range, was achieved upon high-level expression of the Cdr1p, Pdr5p, and Snq2p transporters in an S. cerevisiae strain in which the endogenous interfering activities were further reduced by genetic manipulation. An analysis of a set of therapeutically used and newly synthesized phenothiazine derivatives revealed different pharmacological profiles for Cdr1p, Pdr5p, and Snq2p. All transporters showed similar sensitivities to M961 inhibition. In contrast, Cdr1p was less sensitive to inhibition by fluphenazine, whereas phenothiazine selectively inhibited Snq2p. The inhibition potencies measured by the new assay reflected the ability of the compounds to potentiate the antifungal effect of ketoconazole (KTC), which was detoxified by the overproduced transporters. They also correlated with the 50% inhibitory concentration for inhibition of Pdr5p-mediated transport of rhodamine 6G in isolated plasma membranes. The most active derivative, M961, potentiated the activity of KTC against an azole-resistant CDR1-overexpressing C. albicans isolate.

scholarly journals Identification of Upregulators of BMP2 Expression via High-Throughput Screening of a Synthetic and Natural Compound Library

2009 ◽  
Vol 14 (10) ◽  
pp. 1251-1256 ◽  
Author(s):  
Xue Li ◽  
Jun Yang ◽  
Xiaobo He ◽  
Zhaoyong Yang ◽  
Yan Ding ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Natural Compound ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Compound Library ◽  
Screening Assay ◽  
Morphogenetic Protein ◽  
High Throughput Screening Assay ◽  
High Throughput Assay

Bone morphogenetic protein II (BMP2), a member of the transforming growth factor—β (TGF-β) superfamily, is highly expressed in osteoblasts and is a crucial regulator of osteogenic differentiation . Many observations clearly indicate the high potency of BMP2 as an inducer of osteogenesis, and it may be a novel therapeutic target for diseases associated with bone loss, especially in menopausal and postmenopausal women. To discover new agents that enhance the expression of the mouse BMP2, the authors developed a high-throughput assay to screen a synthetic and natural compound library. The cell-based high-throughput screen was conducted in 96-well plates using the clonal murine calvarial MC3T3-E1 cells. These cells were stably transfected with mouse BMP2 promoter-luciferase and calibrated with the known antiosteoporosis compound genistein. Among 3192 compounds screened, 3 agents (daidzein, formononetin, and 2-Acetyldibenzothiophene) were picked up by the high-throughput screening assay, and those compounds were identified as upregulators of BMP2 expression by real-time quantitative reverse transcription—polymerase chain reaction and flow cytometry. Thus, it is demonstrated that this screening model is useful for identifying lead compounds to treat osteoporosis and maintain bone metabolism balance.

scholarly journals High-Throughput Screening Method of Inhibitors that Block the Interaction between 2 Helical Regions of HIV-1 gp41

2005 ◽  
Vol 10 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Bong-Suk Jin ◽  
Won-Kyu Lee ◽  
Kwangseog Ahn ◽  
Myung Kyu Lee ◽  
Yeon Gyu Yu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Compound Library ◽  
Elisa Method ◽  
Helical Bundle ◽  
Fusion Inhibitors ◽  
Hiv 1

The HIV-1 envelope glycoprotein transmembrane subunit, gp41, mediates the fusion of viral and target cell membranes. The 2 helical regions in the ectodomain of gp41, the N-helix and the C-helix, form a helical bundle complex that has been suggested as a fusion-active conformation. Previously, an enzyme-linked immunosorbent assay (ELISA) method had been established to measure the interaction of 2 helical regions of gp41. In this study, the ELISA method was modified to apply high-throughput screening (HTS) of an organic compound library. A few compounds had been identified to prevent the interaction between 2 helical regions of gp41, and they were further shown to inhibit the gp41-mediated viral infection. In addition, they specifically quenched the fluorescence of tryptophan in the N-helix region, indicating that these compounds bound to the N-helix rather than the C-helix of gp41. These results suggested that this assay method targeting gp41 could be used for HTS of HIV fusion inhibitors. ( Journal of Biomolecular Screening 2005:13-19)

High Throughput Screening Assay to Identify Modulators of IL-17 Expression

2018 ◽  
Vol 20 (9) ◽  
pp. 804-819 ◽  
Author(s):  
Mohamed Boudjelal ◽  
Ana Maria Ruiz-Avendano ◽  
Gonzalo Colmenarejo ◽  
Sergio A. Senar-Sancho ◽  
Ashley Barnes ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay
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