title: A fluorescence polarization based assay for the identification and characterization of polymerase inhibitors.
abstract: A homogenous fluorescence polarization (FP) assay was developed to monitor the enzymatic activity of polymerases. Under the optimized conditions established in this study, the assay provides highly robust and reproducible data. Miniaturization of the assay for high-throughput screening and compound testing was also performed. The sensitivity of the newly developed assay was confirmed using 2',3'-dideoxyadenosine-5'-triphosphate (ddATP), a chain-elongating inhibitor of the polymerase reaction. Side-by-side comparison of the presented fluorescence polarization assay with already well established PicoGreen® fluorescence intensity assay revealed that the performance of both formats is comparable with good assay sensitivity. However, the direct ratiometric readout of the presented FP assay makes it superior over existing colorimetric and fluorescence intensity based assays in terms of susceptibility to false positives. Moreover, due to its generic nature the presented FP assay can be applied to other polymerases and is compatible with identification of inhibitors and requirements of hit-to-lead programs.
authors: Evotec AG, Manfred Eigen Campus, Essener Bogen 7, D-22419 Hamburg, Germany. Electronic address: hamid.nasiri@evotec.com.
authors: Hamid R
authors: HR
authors: Nasiri
authors: Evotec AG, Manfred Eigen Campus, Essener Bogen 7, D-22419 Hamburg, Germany.
authors: László
authors: L
authors: Kiss
authors: Evotec AG, Manfred Eigen Campus, Essener Bogen 7, D-22419 Hamburg, Germany.
authors: Joachim
authors: J
authors: Krämer
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer
chemical_list: D004791:Enzyme Inhibitors
keywords: Assay development; Biochemical assays; Fluorescence polarization; Hit identification; Polymerases