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This version was published on February 1, 2006
Journal of Biomolecular Screening, Vol. 11, No. 1, 75-81 (2006)
DOI: 10.1177/1087057105283296

Utilization of Substrate-Induced Quenching for Screening Targets Promoting NADH and NADPH Consumption

María Jesús Vázquez

Assay Development, Glaxo SmithKline, Madrid, Spain

Stephen Ashman

Assay Development, Glaxo Smith Kline, Essex, United Kingdom

Fernando Ramón

Screening and Compound Profiling, Glaxo Smith Kline, Madrid, Spain

David Calvo

Assay Development, Glaxo Smith Kline, Madrid, Spain

Ana Bardera

J. Julio Martín

Screening and Compound Profiling, Glaxo Smith Kline, Madrid, Spain

Martin Rüdiger

David Tew

Assay Development, Glaxo Smith Kline, Essex, United Kingdom

Juan Manuel Domínguez

Assay Development, Glaxo Smith Kline, Madrid, Spain

Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappearswith oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based onmonitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.

Key Words: substrate-induced quenching • high-throughput screening • NADH • NADPH • dehydrogenase • quenching


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