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

DNA helicases are responsible for the unwinding of double-stranded DNA, fac ilitated by the binding and hydrolysis of 5'-nucleoside triphosphates, Thes e enzymes represent an important class of targets for the development of no vel anti-infective agents particularly because opportunity exists for syner gy with existing therapies targeted at other enzymes involved in DNA replic ation. Unwinding reactions are conventionally monitored by low throughput, gel-based radiochemical assays; to overcome the limitations of low throughp ut to achieve comprehensive characterization of adenosine triphosphate (ATP )-dependent unwinding by viral and bacterial helicases and the screening fo r unwinding inhibitors, we have developed and validated homogeneous time-re solved fluorescence energy transfer (TRET) assays, Rapid characterization a nd screening of DNA helicase has been performed in 96- and 384-well plate d ensities, and the ability to assay in 1536-well format also demonstrated. W e have successfully validated and are running full high throughput runs usi ng 384-well TRET helicase assays, culminating in the identification of a ra nge 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-pos itives due to the relatively high levels of compound quenching (unlike othe r Ln(3+)-based assays). This strategy was successful yet emphasised the nee d for further improvements in helicase assays.