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An Improved ß-Lactamase Reporter Assay: Multiplexing with a Cytotoxicity Readout for Enhanced Accuracy of Hit IdentificationInvitrogen Discovery Sciences, Madison,Wisconsin
Current address, BellBrook Labs, Madison, Wisconsin
Invitrogen Discovery Sciences, Madison,Wisconsin
Invitrogen Discovery Sciences, Madison,Wisconsin
Invitrogen Discovery Sciences, Madison,Wisconsin
Invitrogen Discovery Sciences, Madison,Wisconsin
Invitrogen Discovery Sciences, Madison,Wisconsin, tina.hallis{at}invitrogen.com
A problem inherent to the use of cellular assays for drug discovery is their sensitivity to cytotoxic compounds, which can result in false hits from certain compound screens. To alleviate the need to follow-up hits from a reporter assay with a separate cytotoxicity assay, the authors have developed a multiplexed assay that combines the readout of a ß-lactamase reporter with that of a homogeneous cytotoxicity indicator. Important aspects to the development of the multiplexed format are addressed, including results that demonstrate that the IC50 values of 40 select compounds in a ß-lactamase reporter assay for nuclear factor
Key Words: high-throughput screening (HTS) • Förster resonance energy transfer (FRET) • ß-lactamase • reporter assays • cytotoxicity • multiplex
This version was published on August
1, 2007 Journal of Biomolecular Screening, Vol. 12, No. 5,
635-644 (2007) |
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B and SIE pathway antagonists are not affected by the addition of the cytotoxicity indicator. To demonstrate the improvement in hit confirmation, the multiplexed assay was used to perform a small-library screen (7728 compounds) for serotonin 5HT1A receptor antagonists. Hits identified from analysis of the ß-lactamase reporter data alone were compared to those hits determined when the reporter and cytotoxicity data generated from the multiplexed assay were combined. Confirmation rates were determined from compound follow-up using dose-response analysis of the potential antagonist hits identified by the initial screen. In this representative screen, the multiplexed assay approach yielded a 19% reduction in the number of compounds flagged for follow-up, with a 37% decrease in the number of false hits, demonstrating that multiplexing a ß-lactamase reporter assay with a cytotoxicity readout is a highly effective strategy for reducing false hit rates in cell-based compound screening assays. ( Journal of Biomolecular Screening 2007:635-644)