title: Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening.
abstract: 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.
authors.affiliation: Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Harlow, Essex, United Kingdom.
authors.initials: DL
authors.lastname: Earnshaw
authors.initials: KJ
authors.lastname: Moore
authors.initials: CJ
authors.lastname: Greenwood
authors.initials: H
authors.lastname: Djaballah
authors.initials: AJ
authors.lastname: Jurewicz
authors.initials: KJ
authors.lastname: Murray
authors.initials: AJ
authors.lastname: Pope