Time-resolved fluorescence energy transfer DNA helicase assays for high throughput screening

被引:40
作者
Earnshaw, DL [1 ]
Moore, KJ [1 ]
Greenwood, CJ [1 ]
Djaballah, H [1 ]
Jurewicz, AJ [1 ]
Murray, KJ [1 ]
Pope, AJ [1 ]
机构
[1] SmithKline Beecham Pharmaceut, Mol Screening Technol, Harlow CM19 5AW, Essex, England
关键词
D O I
10.1177/108705719900400505
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 Ln(3+)-based assays). This strategy was successful yet emphasised the need for further improvements in helicase assays.
引用
收藏
页码:239 / 248
页数:10
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