scholarly journals ATP–Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening

2015 ◽  
Vol 88 (1) ◽  
pp. 84-94 ◽  
Author(s):  
Surtaj H. Iram ◽  
Simon J. Gruber ◽  
Olga N. Raguimova ◽  
David D. Thomas ◽  
Seth L. Robia
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Atp Binding ◽  
Fluorescence Energy Transfer ◽  
Atp Binding Cassette Transporter ◽  
Structure Changes ◽  
Fluorescence Energy ◽  
Transporter Structure ◽  
Atp Binding Cassette

Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening

1999 ◽  
Vol 4 (5) ◽  
pp. 239-248 ◽  
Author(s):  
David L. Earnshaw ◽  
Keith J. Moore ◽  
Catherine J. Greenwood ◽  
Hakim Djaballah ◽  
Anthony J. Jurewicz ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dna Helicase ◽  
Fluorescence Energy Transfer ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Dna Helicases ◽  
Rapid Characterization ◽  
Fluorescence Energy

DNA helicases are responsible for the unwinding of double-stranded DNA, facilitated by the binding and hydrolysis of 5′-nucleoside triphosphates. These enzymes represent an important class of targets for the development of novel anti-infective agents particularly because opportunity exists for synergy with existing therapies targeted at other enzymes involved in DNA replication. Unwinding reactions are conventionally monitored by low throughput, gel-based radiochemical assays; to overcome the limitations of low throughput to achieve comprehensive characterization of adenosine triphosphate (ATP)-dependent unwinding by viral and bacterial helicases and the screening for unwinding inhibitors, we have developed and validated homogeneous time-resolved fluorescence energy transfer (TRET) assays. Rapid characterization and screening of DNA helicase has been performed in 96- and 384-well plate densities, and the ability to assay in 1536-well format also demonstrated. We have successfully validated and are running full high throughput runs using 384-well TRET helicase assays, culminating in the identification of a range of chemically diverse inhibitors of viral and bacterial helicases. For screening in mixtures, we used a combination of quench correction routines and confirmatory scintillation proximity (SP) assays to eliminate false-positives due to the relatively high levels of compound quenching (unlike other Ln3+-based assays). This strategy was successful yet emphasised the need for further improvements in helicase assays.

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 A High-Throughput Time-Resolved Fluorescence Energy Transfer Assay to Screen for Modulators of RGS7/Gβ5/R7BP Complex

2018 ◽  
Vol 16 (3) ◽  
pp. 150-161 ◽  
Author(s):  
Brian S. Muntean ◽  
Dipak N. Patil ◽  
Franck Madoux ◽  
James Fossetta ◽  
Louis Scampavia ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
Fluorescence Energy Transfer ◽  
Time Resolved Fluorescence ◽  
Throughput Time ◽  
Time Resolved ◽  
Fluorescence Energy

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.

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